2,2-difluoropropionamide derivatives of bardoxolone methyl, polymorphic forms and methods of use thereof

ABSTRACT

The present invention relates generally to the compound:N-((4aS,6aR,6bS,8aR,12aS,14aR,14bS)-11-cyano-2,2,6a,6b,9,9,12a-heptamethyl-10,14-dioxo-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,12a,14,14a,14b-octadecahydropicen-4a-yl)-2,2-difluoropropanamide,polymorphic forms thereof, methods for preparation and use thereof, pharmaceutical compositions thereof, and kits and articles of manufacture thereof.

This application is a continuation of U.S. patent application Ser. No.16/186,051, filed Nov. 9, 2018, which is a continuation of U.S. patentapplication Ser. No. 15/821,508, filed Nov. 22, 2017, abandoned, whichis a continuation of U.S. patent application Ser. No. 14/260,532, filedApr. 24, 2014, now U.S. Pat. No. 9,856,286, issued on Jan. 2, 2018,which claims priority to U.S. Provisional Application Ser. No.61/815,502 filed Apr. 24, 2013, the entirety of each of which isincorporated herein by reference.

BACKGROUND OF THE INVENTION I. Field of the Invention

The present invention relates generally to the compound:

-   -   N-((4aS,6aR,6bS,8aR,12aS,14aR,14bS)-11-cyano-2,2,6a,6b,9,9,12a-heptamethyl-10,14-dioxo-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,12a,14,14a,14b-octadecahydropicen-4a-yl)-2,2-difluoropropanamide,        also referred to herein as RTA 408, 63415, or PP415. The present        invention also relates to polymorphic forms thereof, methods for        preparation and use thereof, pharmaceutical compositions        thereof, and kits and articles of manufacture thereof.

II. Description of Related Art

The anti-inflammatory and anti-proliferative activity of the naturallyoccurring triterpenoid, oleanolic acid, has been improved by chemicalmodifications. For example,2-cyano-3,12-diooxooleana-1,9(11)-dien-28-oic acid (CDDO) and relatedcompounds have been developed. See Honda et al., 1997; Honda et al.,1998; Honda et al., 1999; Honda et al., 2000a; Honda et al., 2000b;Honda et al., 2002; Suh et al., 1998; Suh et al., 1999; Place et al.,2003; Liby et al., 2005; and U.S. Pat. Nos. 8,129,429, 7,915,402,8,124,799, and 7,943,778, all of which are incorporated herein byreference. The methyl ester, bardoxolone methyl (CDDO—Me), has beenevaluated in phase II and III clinical trials for the treatment andprevention of diabetic nephropathy and chronic kidney disease. SeePergola et al., 2011, which is incorporated herein by reference.

Synthetic triterpenoid analogs of oleanolic acid have also been shown tobe inhibitors of cellular inflammatory processes, such as the inductionby IFN-γ of inducible nitric oxide synthase (iNOS) and of COX-2 in mousemacrophages. See Honda et al, (2000a), Honda et al. (2000b), Honda etal. (2002), and U.S. Pat. Nos. 8,129,429, 7,915,402, 8,124,799, and7,943,778, which are all incorporated herein by reference. Compoundsderived from oleanolic acid have been shown to affect the function ofmultiple protein targets and thereby modulate the activity of severalimportant cellular signaling pathways related to oxidative stress, cellcycle control, and inflammation (e.g., Dinkova-Kostova et al., 2005;Ahmad et al., 2006; Ahmad et al., 2008; Liby et al., 2007, and U.S. Pat.Nos. 8,129,429, 7,915,402, 8,124,799, and 7,943,778).

Given that the biological activity profiles of known triterpenoidderivatives vary, and in view of the wide variety of diseases that maybe treated or prevented with compounds having potent antioxidant andanti-inflammatory effects, and the high degree of unmet medical needrepresented within this variety of diseases, it is desirable tosynthesize new compounds with different biological activity profiles forthe treatment or prevention of one or more indications.

SUMMARY OF THE INVENTION

In some aspects of the present invention, there is provided a compoundof the formula (also referred to as RTA 408, 63415, or PP415):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound is in the form of a pharmaceuticallyacceptable salt. In some embodiments, the compound is not in the form ofa salt.

In another aspect of the present invention, there are providedpolymorphic forms of the above compound.

In some embodiments, the polymorphic form is crystalline, having anX-ray powder diffraction pattern (CuKα) comprising peaks at about10.601, 11.638, 12.121, 13.021, 13,435, 15.418, 15.760, 17.830, 18.753,and 19.671 °2θ. In some embodiments, the X-ray powder diffractionpattern (CuKα) is substantially as shown in FIG. 53.

In some embodiments, the polymorphic form is crystalline, having anX-ray powder diffraction pattern (CuKα) comprising peaks at about 7.552,10.339, 11.159, 12.107, 14.729, 15.329, 15.857, 16.824, 17.994, 18.344,19.444, 19.764, 20.801, and 22.414 °2θ. In some embodiments, the X-raydiffraction pattern (CuKα) is substantially as shown in FIG. 56.

In another aspect of the present invention, there are providedpharmaceutical compositions comprising an active ingredient consistingof the above compound or polymorphic forms thereof (such as, e.g., anyone of the polymorphic forms described herein above or below), and apharmaceutically acceptable carrier. In some embodiments, thepharmaceutical composition is formulated for administration: orally,intraadiposally, intraarterially, intraarticularly, intracranially,intradermally, intralesionally, intramuscularly, intranasally,intraocularly, intrapericardially, intraperitoneally, intrapleurally,intraprostatically, intrarectally, intrathecally, intratracheally,intratumorally, intraumbilically, intravaginally, intravenously,intravesicularlly, intravitreally, liposomally, locally, mucosally,parenterally, rectally, subconjunctival, subcutaneously, sublingually,topically, transbuccally, transdermally, vaginally, in crémes, in lipidcompositions, via a catheter, via a lavage, via continuous infusion, viainfusion, via inhalation, via injection, via local delivery, or vialocalized perfusion. In some embodiments, the pharmaceutical compositionis formulated for oral, intraarterial, intravenous, or topicaladministration. In some embodiments, the pharmaceutical composition isformulated for oral administration.

In some embodiments, the pharmaceutical composition is formulated as ahard or soft capsule, a tablet, a syrup, a suspension, a soliddispersion, a wafer, or an elixir. In some embodiments, thepharmaceutical composition according to the invention further comprisesan agent that enhances solubility and dispersibility. In someembodiments, the compound or polymorphic form is suspended in sesameoil.

In other embodiments, the pharmaceutical composition is formulated fortopical administration. In other embodiments, the pharmaceuticalcomposition is formulated as a lotion, a cream, a gel, an oil, anointment, a salve, or a suspension. In some embodiments, thepharmaceutical composition is formulated as a lotion, as a cream, or asa gel. In some embodiments, the amount of the active ingredient is fromabout 0.01% to about 5% by weight, about 0.01% to about 3% by weight, or0.01%, 0.1%, 1%, or 3% by weight.

In another aspect of the present invention there are provided methods oftreating or preventing a condition associated with inflammation oroxidative stress in a patient in need thereof, comprising administeringto the patient a therapeutically effective amount of the pharmaceuticalcomposition as described above or below. The invention likewise relatesto the compoundN-((4aS,6aR,6bS,8aR,12aS,14aR,14bS)-11-cyano-2,2,6a,6b,9,9,12a-heptamethyl-10,14-dioxo-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,12a,14,14a,14b-octadecahydropicen-4a-yl)-2,2-difluoropropanamide(or RTA 408, 63415, or PP415) or a pharmaceutically acceptable saltthereof, or a polymorphic form of that compound (such as, e.g., any oneof the polymorphic forms described herein above or below), or apharmaceutical composition comprising any of the aforementioned entitiesand a pharmaceutically acceptable carrier (including, e.g., thepharmaceutical compositions described above), for use in treating orpreventing a condition associated with inflammation or oxidative stress.The invention also relates to the use of the aforementioned compound,polymorphic form or pharmaceutical composition for the preparation of amedicament for the treatment or prevention of a condition associatedwith inflammation or oxidative stress. In some embodiments, thecondition is associated with inflammation. In other embodiments, thecondition is associated with oxidative stress. In some embodiments, thecondition is a skin disease or disorder, sepsis, dermatitis,osteoarthritis, cancer, inflammation, an autoimmune disease,inflammatory bowel disease, a complication from localized or total-bodyexposure to ionizing radiation, mucositis, acute or chronic organfailure, liver disease, pancreatitis, an eye disorder, a lung disease ordiabetes.

The present invention furthermore relates to the compoundN-((4aS,6aR,6bS,8aR,12aS,14aR,14bS)-11-cyano-2,2,6a,6b,9,9,12a-heptamethyl-10,14-dioxo-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,12a,14,14a,14b-octadecahydropicen-4a-yl)-2,2-difluoropropanamide(or RTA 408) or a pharmaceutically acceptable salt thereof, or apolymorphic form of that compound (such as, e.g., any one of thepolymorphic forms described herein above or below), or a pharmaceuticalcomposition comprising any of the aforementioned entities and apharmaceutically acceptable carrier (including, e.g., the pharmaceuticalcompositions described above), for use in treating or preventing acondition selected from a skin disease or disorder, sepsis, dermatitis,osteoarthritis, cancer, inflammation, an autoimmune disease,inflammatory bowel disease, a complication from localized or total-bodyexposure to ionizing radiation, mucositis, acute or chronic organfailure, liver disease, pancreatitis, an eye disorder, a lung disease,or diabetes. Accordingly, the invention relates to the use of theaforementioned compound, polymorphic form or pharmaceutical compositionfor the preparation of a medicament for the treatment or prevention of acondition selected from a skin disease or disorder, sepsis, dermatitis,osteoarthritis, cancer, inflammation, an autoimmune disease,inflammatory bowel disease, a complication from localized or total-bodyexposure to ionizing radiation, mucositis, acute or chronic organfailure, liver disease, pancreatitis, an eye disorder, a lung disease,or diabetes. The invention also relates to a method of treating orpreventing a condition selected from a skin disease or disorder, sepsis,dermatitis, osteoarthritis, cancer, inflammation, an autoimmune disease,inflammatory bowel disease, a complication from localized or total-bodyexposure to ionizing radiation, mucositis, acute or chronic organfailure, liver disease, pancreatitis, an eye disorder, a lung disease,or diabetes in a patient in need thereof, the method comprisingadministering to the patient a therapeutically effective amount of theaforementioned compound, polymorphic form or pharmaceutical composition.

In some embodiments, the condition is a skin disease or disorder such asdermatitis, a thermal or chemical burn, a chronic wound, acne, alopecia,other disorders of the hair follicle, epidermolysis bullosa, sunburn,complications of sunburn, disorders of skin pigmentation, anaging-related skin condition; a post-surgical wound, a scar from a skininjury or burn, psoriasis, a dermatological manifestation of anautoimmune diseases or a graft-versus host disease, skin cancer; or adisorder involving hyperproliferation of skin cells. In someembodiments, the skin disease or disorder is dermatitis. In someembodiments, the dermatitis is allergic dermatitis, atopic dermatitis,dermatitis due to chemical exposure, or radiation-induced dermatitis. Inother embodiments, the skin disease or disorder is a chronic wound. Insome embodiments, the chronic wound is a diabetic ulcer, a pressuresore, or a venous ulcer. In other embodiments, the skin disease ordisorder is alopecia. In some embodiments, the alopecia is selected frombaldness or drug-induced alopecia. In other embodiments, the skindisease or disorder is a disorder of skin pigmentation. In someembodiments, the disorder of skin pigmentation is vitiligo. In otherembodiments, the skin disease or disorder is a disorder involvinghyperproliferation of skin cells. In some embodiments, the disorderinvolving hyperproliferation of skin cells is hyperkeratosis.

In other embodiments, the condition is an autoimmune disease, such asrheumatoid arthritis, lupus, Crohn's disease, or psoriasis. In otherembodiments, the condition is liver disease, such as fatty liver diseaseor hepatitis.

In other embodiments, the condition is an eye disorder, such as uveitis,macular degeneration, glaucoma, diabetic macular edema, blepharitis,diabetic retinopathy, a disease or disorder of the corneal endothelium,post-surgical inflammation, dry eye, allergic conjunctivitis or a formof conjunctivitis. In some embodiments, the eye disorder is maculardegeneration. In some embodiments, the macular degeneration is the dryform. In other embodiments, the macular degeneration is the wet form. Insome embodiments, the disease or disorder of the corneal endothelium isFuchs endothelial corneal dystrophy.

In other embodiments, the condition is a lung disease, such as pulmonaryinflammation, pulmonary fibrosis, COPD, asthma, cystic fibrosis, oridiopathic pulmonary fibrosis. In some embodiments, the COPD is inducedby cigarette smoke.

In other embodiments, the condition is sepsis. In other embodiments, thecondition is mucositis resulting from radiation therapy or chemotherapy.In some embodiments, the mucositis presents orally. In otherembodiments, the condition is associated with exposure to radiation. Insome embodiments, the radiation exposure leads to dermatitis. In someembodiments, the radiation exposure is acute. In other embodiments, theradiation exposure is fractionated.

In other embodiments, the condition is cancer. In some non-limitingembodiments, the cancer is leukemia, lymphoma, multiple myeloma, orcancer of the breast, skin, lung, pancreas, liver, stomach, smallintestine, large intestine or colon, gall bladder, esophagus, ovary,endometrium, cervix, oral or nasal mucosa, brain, prostate, bladder,urogenital tract, testicle, kidney, genitalia, thyroid, or muscletissue. In some embodiments, the cancer is a carcinoma or sarcoma.

In some embodiments, the compound or composition of the invention isadministered before or immediately after a subject is treated withradiation therapy, chemotherapy, or both. In some embodiments, thecompound or composition of the invention is administered both before andafter the subject is treated with radiation therapy, chemotherapy orboth. In some embodiments, the effect of the composition of theinvention is to reduce side effects of radiation therapy, chemotherapy,or combined radio- and chemo-therapy, including mucositis anddermatitis. In some embodiments, the effect of the composition of theinvention is to enhance the efficacy of the radiation therapy,chemotherapy, or combined radio- and chemo-therapy. In some embodiments,the effect of the composition of the invention is to reduce the sideeffects of, and enhance the efficacy of, the radiation therapy,chemotherapy, or combined radio- and chemo-therapy.

Combination treatment therapy is also contemplated by the presentdisclosure. For example, regarding methods of treating cancer in asubject, comprising administering to the subject a pharmaceuticallyeffective amount of a compound of the present disclosure, the method mayfurther comprise a treatment selected from the group consisting ofadministering a pharmaceutically effective amount of a second drug,radiotherapy, gene therapy, and surgery. Such methods may furthercomprise (1) contacting a tumor cell with the compound prior tocontacting the tumor cell with the second drug, (2) contacting a tumorcell with the second drug prior to contacting the tumor cell with thecompound, or (3) contacting a tumor cell with the compound and thesecond drug at the same time. The second drug may, in certainembodiments, be an antibiotic, anti-inflammatory, anti-neoplastic,anti-proliferative, anti-viral, immunomodulatory, or immunosuppressive.The second drug may be an alkylating agent, androgen receptor modulator,cytoskeletal disruptor, estrogen receptor modulator, histone-deacetylaseinhibitor, HMG-CoA reductase inhibitor, prenyl-protein transferaseinhibitor, retinoid receptor modulator, topoisomerase inhibitor, ortyrosine kinase inhibitor. In certain embodiments, the second drug is5-azacitidine, 5-fluorouracil, 9-cis-retinoic acid, actinomycin D,alitretinoin, all-trans-retinoic acid, annamycin, axitinib, belinostat,bevacizumab, bexarotene, bosutinib, busulfan, capecitabine, carboplatin,carmustine, CD437, cediranib, cetuximab, chlorambucil, cisplatin,cyclophosphamide, cytarabine, dacarbazine, dasatinib, daunorubicin,decitabine, docetaxel, dolastatin-10, doxifluridine, doxorubicin,doxorubicin, epirubicin, erlotinib, etoposide, etoposide, gefitinib,gemcitabine, gemtuzumab ozogamicin, hexamethylmelamine, idarubicin,ifosfamide, imatinib, irinotecan, isotretinoin, ixabepilone, lapatinib,LBH589, lomustine, mechlorethamine, melphalan, mercaptopurine,methotrexate, mitomycin, mitoxantrone, MS-275, neratinib, nilotinib,nitrosourea, oxaliplatin, paclitaxel, plicamycin, procarbazine,semaxanib, semustine, sodium butyrate, sodium phenylacetate,streptozotocin, suberoylanilide hydroxamic acid, sunitinib, tamoxifen,teniposide, thiopeta, tioguanine, topotecan, TRAIL, trastuzumab,tretinoin, trichostatin A, valproic acid, valrubicin, vandetanib,vinblastine, vincristine, vindesine, or vinorelbine.

Methods of treating or preventing a disease with an inflammatorycomponent in a subject, comprising administering to the subject apharmaceutically effective amount of a compound of the presentdisclosure are also contemplated. The disease may be, for example, lupusor rheumatoid arthritis. The disease may be an inflammatory boweldisease, such as Crohn's disease or ulcerative colitis. The disease withan inflammatory component may be a cardiovascular disease. The diseasewith an inflammatory component may be diabetes, such as type 1 or type 2diabetes. RTA 408 may also be used to treat complications associatedwith diabetes. Such complications are well-known in the art and include,for example, obesity, hypertension, atherosclerosis, coronary heartdisease, stroke, peripheral vascular disease, hypertension, nephropathy,neuropathy, myonecrosis, retinopathy and metabolic syndrome (syndromeX). The disease with an inflammatory component may be a skin disease,such as psoriasis, acne, or atopic dermatitis. Administration of a RTA408 in treatment methods of such skin diseases may be, for example,topical or oral.

The disease with an inflammatory component may be metabolic syndrome(syndrome X). A patient having this syndrome is characterized as havingthree or more symptoms selected from the following group of fivesymptoms: (1) abdominal obesity; (2) hypertriglyceridemia; (3) lowhigh-density lipoprotein cholesterol (HDL); (4) high blood pressure; and(5) elevated fasting glucose, which may be in the range characteristicof Type 2 diabetes if the patient is also diabetic. Each of thesesymptoms is defined in the Third Report of the National CholesterolEducation Program Expert Panel on Detection, Evaluation and Treatment ofHigh Blood Cholesterol in Adults (Adult Treatment Panel III, or ATPIII), National Institutes of Health, 2001, NIH Publication No. 01-3670,incorporated herein by reference. Patients with metabolic syndrome,whether or not they have or develop overt diabetes mellitus, have anincreased risk of developing the macrovascular and microvascularcomplications that are listed above that occur with type 2 diabetes,such as atherosclerosis and coronary heart disease.

Another general method of the present disclosure entails a method oftreating or preventing a cardiovascular disease in a subject, comprisingadministering to the subject a pharmaceutically effective amount of acompound of the present disclosure. The cardiovascular disease may be,for example, atherosclerosis, cardiomyopathy, congenital heart disease,congestive heart failure, myocarditis, rheumatic heart disease, valvedisease, coronary artery disease, endocarditis, or myocardialinfarction. Combination therapy is also contemplated for such methods.For example, such methods may further comprise administering apharmaceutically effective amount of a second drug. The second drug maybe, for example, a cholesterol lowering drug, an anti-hyperlipidemic, acalcium channel blocker, an anti-hypertensive, or an HMG-CoA reductaseinhibitor. Non-limiting examples of second drugs include amlodipine,aspirin, ezetimibe, felodipine, lacidipine, lercanidipine, nicardipine,nifedipine, nimodipine, nisoldipine or nitrendipine. Other non-limitingexamples of second drugs include atenolol, bucindolol, carvedilol,clonidine, doxazosin, indoramin, labetalol, methyldopa, metoprolol,nadolol, oxprenolol, phenoxybenzamine, phentolamine, pindolol, prazosin,propranolol, terazosin, timolol or tolazoline. The second drug may be,for example, a statin, such as atorvastatin, cerivastatin, fluvastatin,lovastatin, mevastatin, pitavastatin, pravastatin, rosuvastatin orsimvastatin.

Methods of treating or preventing a neurodegenerative disease in asubject, comprising administering to the subject a pharmaceuticallyeffective amount of a compound of the present disclosure are alsocontemplated. The neurodegenerative disease may, for example, beselected from the group consisting of Parkinson's disease, Alzheimer'sdisease, multiple sclerosis (MS), Huntington's disease and amyotrophiclateral sclerosis. In particular embodiments, the neurodegenerativedisease is Alzheimer's disease. In particular embodiments, theneurodegenerative disease is MS, such as primary progressive,relapsing-remitting secondary progressive or progressive relapsing MS.The subject may be, for example, a primate. The subject may be a human.

In particular embodiments of methods of treating or preventing aneurodegenerative disease in a subject, comprising administering to thesubject a pharmaceutically effective amount of a compound of the presentdisclosure, the treatment suppresses the demyelination of neurons in thesubject's brain or spinal cord. In certain embodiments, the treatmentsuppresses inflammatory demyelination. In certain embodiments, thetreatment suppresses the transection of neuron axons in the subject'sbrain or spinal cord. In certain embodiments, the treatment suppressesthe transection of neurites in the subject's brain or spinal cord. Incertain embodiments, the treatment suppresses neuronal apoptosis in thesubject's brain or spinal cord. In certain embodiments, the treatmentstimulates the remyelination of neuron axons in the subject's brain orspinal cord. In certain embodiments, the treatment restores lostfunction after an MS attack. In certain embodiments, the treatmentprevents a new MS attack. In certain embodiments, the treatment preventsa disability resulting from an MS attack.

One general aspect of the present disclosure contemplates a method oftreating or preventing a disorder characterized by overexpression ofiNOS genes in a subject, comprising administering to the subject apharmaceutically effective amount of a compound of the presentdisclosure.

Another general aspect of the present disclosure contemplates a methodof inhibiting IFN-γ-induced nitric oxide production in cells of asubject, comprising administering to said subject a pharmaceuticallyeffective amount of a compound of the present disclosure.

Yet another general method of the present disclosure contemplates amethod of treating or preventing a disorder characterized byoverexpression of COX-2 genes in a subject, comprising administering tothe subject a pharmaceutically effective amount of compound of thepresent disclosure.

Methods of treating renal/kidney disease (RKD) in a subject, comprisingadministering to the subject a pharmaceutically effective amount of acompound of the present disclosure are also contemplated. See U.S.patent application Ser. No. 12/352,473, which is incorporated byreference herein in its entirety. The RKD may result from, for example,a toxic insult. The toxic insult may result from, for example, animaging agent or a drug. The drug may be a chemotherapeutic, forexample. The RKD may result from ischemia/reperfusion injury, in certainembodiments. In certain embodiments, the RKD results from diabetes orhypertension. The RKD may result from an autoimmune disease. The RKD maybe further defined as chronic RKD, or acute RKD.

In certain methods of treating renal/kidney disease (RKD) in a subject,comprising administering to the subject a pharmaceutically effectiveamount of a compound of the present disclosure, the subject hasundergone or is undergoing dialysis. In certain embodiments, the subjecthas undergone or is a candidate to undergo kidney transplant. Thesubject may be a primate. The primate may be a human. The subject inthis or any other method may be, for example, a cow, horse, dog, cat,pig, mouse, rat or guinea pig.

Also contemplated by the present disclosure is a method for improvingglomerular filtration rate or creatinine clearance in a subject,comprising administering to the subject a pharmaceutically effectiveamount of a compound of the present disclosure.

In some embodiments, the pharmaceutical composition is administered in asingle dose per day. In other embodiments, the pharmaceuticalcomposition is administered in more than one dose per day. In someembodiments, the pharmaceutical composition is administered in apharmaceutically effective amount.

In some embodiments, the active ingredient is administered in a dosefrom about 1 mg/kg to about 2000 mg/kg. In other embodiments, the doseis from about 3 mg/kg to about 100 mg/kg. In other embodiments, the doseis about 3, 10, 30, or 100 mg/kg.

In other embodiments, the pharmaceutical composition is administeredtopically. In some embodiments, the topical administration isadministered to the skin. In other embodiments, the topicaladministration is administered to the eye.

In other embodiments, the pharmaceutical composition is administeredorally. In other embodiments, the pharmaceutical composition isadministered intraocularly.

Other objects, features and advantages of the present disclosure willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating specific embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.Note that simply because a particular compound is ascribed to oneparticular generic formula does not mean that it cannot also belong toanother generic formula.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentdisclosure. The invention may be better understood by reference to oneof these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1—Effect of RTA 408 on IFNγ-induced nitric oxide production andcell viability in RAW264.7 cells.

FIGS. 2a & b—Effect of RTA 408 on antioxidant response elementactivation: (a) NQO1-ARE luciferase activity; (b) GSTA2-ARE luciferaseactivity.

FIGS. 3a-d —Effect of RTA 408 on Nrf2 target gene expression in HFL1lung fibroblasts: (a) NQO1; (b) HMOX1; (c) GCLM; (d) TXNRD1.

FIGS. 4a-d —Effect of RTA 408 on Nrf2 target gene expression in BEAS-2Bbronchial epithelial cells: (a) NQO1; (b) HMOX1; (c) GCLM; (d) TXNRD1.

FIGS. 5a & b—Effect of RTA 408 on Nrf2 target protein levels: (a)SH-SY5Y cells; (b) BV2 cells.

FIG. 6—Effect of RTA 408 on NQO1 enzymatic activity in RAW264.7 cells.

FIG. 7—Effect of RTA 408 on total glutathione levels in the AML-12hepatocyte cell line.

FIG. 8—Effect of RTA 408 on WST-1 absorbance as a marker of NADPH.

FIGS. 9a-d —Effect of RTA 408 on expression of genes involved in NADPHsynthesis: (a) H6PD; (b) PGD; (c) TKT; (d) ME1.

FIG. 10—Effect of RTA 408 on TNFα-induced activation of a NF-κBluciferase reporter construct.

FIG. 11—Effect of RTA 408 on TNFα-induced phosphorylation of IκBα.

FIGS. 12a-d —Effect of RTA 408 on transaminase gene expression: (a) ALT1(GPT1); (b) ALT2 (GPT2); (c) AST1 (GOT1); (d) AST1 (GOT2). Asterisksindicate a statistically-significant difference from the control group(*P<0.05; **P<0.01).

FIG. 13—Effect of RTA 408 on pyruvate levels in cultured muscle cells(*P<0.05).

FIG. 14—RTA 408 activity in a model of pulmonary LPS-mediatedinflammation (% change in pro-inflammatory cytokines relative to LPStreatment). RTA 408 was administered QD×3 at Time 0, 24, and 48 hoursfollowed by LPS one hour after the last dose of RTA 408 in female BALB/cmice. Animals were sacrificed 20 hours after LPS administration. BALFwas examined for pro-inflammatory cytokine expression. RTA 408 reducedpro-inflammatory cytokines: Dose-dependent reductions were observed,with peak reductions ranging from 50%-80% in TNF, IL-6, and IL-12.

FIGS. 15a & b—Effect of RTA 408 on LPS-induced pulmonary inflammation inmice: (a) inflammatory cytokines; (b) Nrf2 targets. Methods: RTA 408administered to female BALB/c mice (n=10) QD×6 at Time 0, 24, 48, 72,96, and 120 hours followed by LPS at 121 hours with animals sacrificedat 141 hours. Pro-inflammatory cytokine protein expression assayed inBALF; Nrf2 biomarkers assayed in lung. Asterisks indicate astatistically significant difference from the saline control group(*P<0.05; **P<0.01; ***P<0.001).

FIGS. 16a & b—RTA 408 reduces BALF infiltrates in bleomycin-inducedpulmonary inflammation: (a) BAL fluid cell count; (b) body weight. RTA408 was administered QD×39 on Days −10 to 28 in C57BL/6 mice. Bleomycinwas given on Day 0. Daily weights were measured. BAL fluid cell countswere obtained at sacrifice. A notable reduction in inflammatoryinfiltrate was observed. No significant improvement in chronicinflammation score, interstitial fibrosis, or number of fibrotic fociwas observed.

FIGS. 17a & b—Effect of RTA 408 on bleomycin-induced pulmonary fibrosisin rats: (a) PMN; (b) Hydroxyproline. Asterisks indicate a statisticallysignificant difference from the bleomycin control group (*P<0.05).

FIG. 18—Effect of RTA 408 on Nrf2 target enzymes in lungs from rats withbleomycin-induced pulmonary fibrosis. Asterisks indicate a statisticallysignificant difference from the saline control group (*P<0.05; **P<0.01;***P<0.001).

FIGS. 19a-e —Effect of RTA 408 on cigarette smoke-induced COPD in mice:(a) KC; (b) IL-6; (c) TNF-α; (d) IFN-γ; (e) RANTES. RTA 408 (63415) wastested at dose levels of 3 mg/kg (low), 10 mg/kg (mid), and 30 mg/kg(high). An AIM analog (63355) was tested in the same study forcomparison. Asterisks indicate a statistically significant differenceform the CS control group.

FIG. 20—Effect of RTA 408 on Nrf2 target enzymes in lungs from mice withcigarette smoke-induced COPD. Asterisks indicate a statisticallysignificant difference from the saline control group (*P<0.05; **P<0.01;***P<0.001). Daggers represent a statistically significant differencefrom mice expose to cigarette smoke and administered vehicle (†P<0.05).

FIGS. 21a-d —show body weight as a function of time of 63415-treatedBALB/c mice that serves as a model of sepsis. LPS was administered toall animals on Day 0. (a) Body Weight: 63415, (b) Body Weight: RTA 405,(c) Systemic LPS: % Survival: 63415, (d) Systemic LPS: % Survival: RTA405. Both RTA 408 and 63415 was administered QD×5 on Days −2 to 2. 63415improved survival.

FIG. 22—RTA 408 activity in a model of radiation-induced oral mucositis.RTA 405 or RTA 408 (63415) was administered BID×20 on Days −5 to −1 andDays 1 to 15 to male Syrian Golden Hamsters. Radiation occurred on Day0. Mucositis scores range from 0 to 5 based on clinical manifestations(0: completely healthy; 1-2: light to severe erythema; 3-5: varyingdegrees of ulceration). RTA 408 (63415) meaningfully improved mucositisat 30 mg/kg and 100 mg/kg with up to a 36% reduction in ulceration.

FIG. 23—Nrf2 target gene induction consistent from RTA 408 (63415)14-day mouse toxicity study in C57BL/6 mice. mRNA of Nrf2 target genesassessed in livers of mice treated PO QD×14. Substantial increases inmRNA expression for multiple Nrf2 target genes were observed and wereconsistent with tissue exposure.

FIGS. 24a & b—Induction of Nrf2 target genes in the rat liver by RTA 408(63415): (a) Target genes; (b) Negative regulators. mRNA of Nrf2 targetgenes was assessed in livers of rats treated PO QD×14.

FIGS. 25a & b—RTA 408 (63415) induces Nrf2 target genes in monkeytissues: (a) Liver; (b) Lung. mRNAs of Nrf2 target genes were assessedin monkeys treated PO QD×14 using Panomics QUANTIGENE® 2.0 Plextechnology.

FIGS. 26a & b—RTA 408 (63415) induces Nrf2 target enzyme activity in themouse liver: (a) NQO1 activity; (b) GST activity. Nrf2 target enzymeactivity was assessed in livers of mice treated PO QD×14. NQO1 and GSTenzyme activities were induced in a dose dependent manner.

FIGS. 27a & b—Induction of target enzyme activity in the rat liver byRTA 408 (63415): (a) NQO1; (b) GST. Nrf2 target enzyme activity wasassessed in livers of rats treated PO QD×14. NQO1 and GST enzymeactivities were induced dose-dependently.

FIGS. 28a & b—RTA 408 (63415) induces Nrf2 target enzyme activity invarious tissues of cynomolgus monkeys: (a) NQO1 activity; (b) GSRactivity.

FIGS. 29a & b—RTA 408 concentration in mouse liver, lung, and brain, andNQO1 activity in mouse liver after 14 days of daily oral administration.(a) Tissue distribution of RTA 408 in mice after 14 days of daily oraladministration. Data represent mean±SD RTA 408 concentrations in tissuecollected 4 hours after the final dose of the study. Numbers above theerror bars are representative of the mean. (b) Correlation of mouseliver RTA 408 content with NQO1 enzyme activity. Individual mouse liverRTA 408 liver content was plotted against individual enzyme activityfrom this report.

FIGS. 30a & b—RTA 408 concentration in rat plasma, liver, lung, andbrain, and NQO1 activity in rat liver after 14 days of daily oraladministration. (a) Tissue distribution of RTA 408 in rats after 14 daysof daily oral administration. Data represent mean±SD RTA 408concentrations in tissue collected 4 hours after the final dose of thestudy. Numbers above the error bars are representative of the mean. *Twovalues were excluded from the mean calculation due to being outliers,defined as values causing the set of data to fail the Shapiro-Wilknormality test. (b) Correlation of rat liver RTA 408 content with NQO1enzyme activity. Individual rat liver RTA 408 liver content was plottedagainst individual enzyme activity from this report. The tissues fromthe 100 mg/kg RTA 408 dose group were collected on Day 6, and theobserved toxicities in this group precluded liver NQO1 enzyme activityevaluations.

FIGS. 31a & b—RTA 408 (63415) treatment of monkeys activated Nrf2 inPBMC cells: (a) PBMC NQO1 vs. Plasma Concentration; (b) Lung NQO1 vs.PBMC NQO1.

FIG. 32—Summary of RTA 408 (63415) 14-day monkey toxicity study. Alldoses were well-tolerated without adverse clinical signs. Clinicalchemistry data suggested no obvious toxicity.

FIG. 33—Plasma concentration of RTA 408 after topical ocular and oraladministrations at different times after dosing. The plasmaconcentration of RTA 408 was also measured after 5 days of daily topicalocular administration of RTA 408 and determined to remain relativelyconsistent from the measurements taken after the first day.

FIGS. 34a & b—Correlation of exposure to RTA 408 in monkey plasma withNQO1 and SRXN1 mRNA expression in PBMCs: (a) NQO1; (b) SRXN1.

FIG. 35—Concentration of RTA 408 in various different tissues or fluidswithin the eye as a function of time after 5 days of topical oculardosing. RTA 408 concentration in plasma was also measured after topicalocular administration.

FIG. 36—Effect of RTA 408 on the incidence of grade 3 dermatitis causedby acute radiation exposure for different concentrations of RTAadministered topically.

FIG. 37—Effect of RTA 408 on the incidence of grade 2 dermatitis overthe course of 30 days caused by acute radiation exposure for differentconcentrations of RTA administered topically.

FIG. 38—Effect of RTA 408 on the incidence of grade 3 dermatitis overthe course of 28 days caused by acute radiation exposure for differentconcentrations of RTA administered orally.

FIG. 39a & b—a) An area under the curve analysis of clinical score ofthe dermatitis as a function of time for each of the different controlgroups including all of the animals used in the test. b) An area underthe curve analysis of the clinical score of the dermatitis as a functionof the duration of that score for each of the different control groupsincluding only animals that completed the entire 30 days in the trial.

FIG. 40—Average 1^(st) blind score of the acute radiation dermatitis asa function of time for untreated, untreated with no radiation exposure,vehicle only and three oral amounts of RTA 408 at 3, 10 and 30 mg/kg.The dermatitis score is based upon the scale that 0 is completelyhealthy, 1-2 exhibits mild to moderate erythema with minimal to slightdesquamation, 3-4 exhibits moderate to severe erythema and desquamation,and 5 exhibits a frank ulcer.

FIG. 41—Mean score of the acute radiation dermatitis as a function oftime for untreated, untreated with no radiation exposure, vehicle onlyand three oral amounts of RTA 408 at 3, 10 and 30 mg/kg measured everyother day from day 4 to day 30. The dermatitis score is based upon thescale that 0 is completely healthy, 1-2 exhibits mild to moderateerythema with minimal to slight desquamation, 3-4 exhibits moderate tosevere erythema and desquamation, and 5 exhibits a frank ulcer.

FIG. 42—Mean score of the acute radiation dermatitis as a function oftime for untreated, untreated with no radiation exposure, vehicle onlyand three topical amounts of RTA 408 at 0.01, 0.1 and 1% measured everyother day from day 4 to day 30. The dermatitis score is based upon thescale that 0 is completely healthy, 1-2 exhibits mild to moderateerythema with minimal to slight desquamation, 3-4 exhibits moderate tosevere erythema and desquamation, and 5 exhibits a frank ulcer.

FIG. 43—Clinical scores of fractional radiation dermatitis plottedversus time and shows the change in dermatitis score for each testinggroup. The scale includes a dermatitis score from 0 to 5 where 0 iscompletely healthy, 1-2 indicates mild to moderate erythema with minimalto slight desquamation, 3-4 indicates moderate to severe erythema anddesquamation, and 5 is a frank ulcer.

FIG. 44—Graph of the AUC analysis showing the dermatitis score(severity * days) for each of the testing groups over the entireobservation period. The dermatitis scores were assessed every two daysfrom day 4 to day 30 of the study.

FIG. 45—Reduction of aqueous humor protein concentrations for differentformulations of RTA 408 (dark bars) compared to literature values forMAXIDEX® (0.1% dexamethasone) and mapracorat (light bars) afterinduction of paracentesis.

FIG. 46—RTA 408 (63415) dose-dependently suppresses NO in vivo. CD-1mice (n=6) were dosed with dimethyl sulfoxide or AIM by oral gavage. LPS(5 mg/kg) was administered 24 h later. Twenty-four hours after LPSadministration, whole blood was collected for NO assay. NO inhibitionwas determined by Griess Reaction from reduced, de-proteinated plasma.

FIG. 47—RTA 408 (63415) distributes extensively into mouse tissues. Micewere dosed with 25 mg/kg PO QD×3 of either RTA 408 (63415) or RTA 405.Blood (plasma and whole blood) and tissues (brain, liver, lung, andkidney) were collected 6 hours after the last dose. Semi-quantitativeanalysis of drug content was performed. Notable levels were observed inthe CNS.

FIG. 48—RTA 408 (63415) induces NQO1 activity in mouse liver, lung, andkidney. Mice were dosed with 25 mg/kg PO QD×3, tissues were collected 6hours after the last dose, and analysis of NQO1 activity was performed.Meaningful activation of NQO1 was observed in multiple tissues.

FIG. 49—Summary of RTA 408 (63415) 14-day mouse toxicity study. C57BL/6mice were dosed PO QD×14. Endpoints included survival, weight, andclinical chemistries. All animals survived to day 14. No significantweight changes occurred compared to the vehicle group, and there was noevidence of toxicity at any dose based on clinical chemistries.

FIG. 50—Tissue distribution from RTA 408 (63415) 14-day mouse toxicitystudy in C57BL/6 mice. Brain, lung, and liver: Collected 4 hours afterfinal dose, quantified for RTA 408 (63415) content using sensitiveLC/MS/MS method. Exposures at 10 and 100 mg/kg: in lung exceeded invitro IC₅₀ for NO induction by 55- and 1138-fold, respectively, and inbrain exceeded in vitro IC₅₀ for NO induction by 29- and 541-fold,respectively.

FIG. 51—RTA 408 (63415) tissue distribution in Sprague Dawley rats. RTA408 (63415) distributes well into target tissues. Tissues were collectedfour hours after final dose on Day 14 or Day 6 (100 mg/kg), extracted,and quantified for RTA 408 (63415) content using a sensitive LC/MS/MSmethod. Exposures at 10 mg/kg in lung and brain exceed in vitro IC₅₀ forNO inhibition by 294- and 240-fold, respectively.

FIG. 52—RTA 408 (63415) target tissue distribution in cynomolgusmonkeys. Tissues were collected four hours after final dose on Day 14.RTA 408 (63415) content was extracted and quantified using a sensitiveLC/MS/MS method.

FIG. 53—PXRD patterns (2-30 °2θ) of RTA 408 Form A.

FIG. 54—DSC thermogram (25-280° C.) of RTA 408 Form A.

FIG. 55—TGA-MS thermogram (25-200° C.) of RTA 408 Form A.

FIG. 56—PXRD patterns (2-30 °2θ) of RTA 408 Form B.

FIG. 57—DSC thermogram (25-280° C.) of RTA 408 Form B.

FIG. 58—TGA-MS thermogram (25-200° C.) of RTA 408 Form B.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The present invention provides in one aspect the compound:

-   -   N-((4aS,6aR,6bS,8aR,12aS,14aR,14bS)-11-cyano-2,2,6a,6b,9,9,12a-heptamethyl-10,14-dioxo-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,12a,14,14a,14b-octadecahydropicen-4a-yl)-2,2-difluoropropanamide,        which is also referred to herein as RTA 408. In other        non-limiting aspects, the present invention also provides        polymorphic forms thereof, including solvates thereof. In other        non-limiting aspects, the invention also provides        pharmaceutically acceptable salts thereof. In other non-limiting        aspects, there are also provided methods for preparation,        pharmaceutical compositions, and kits and articles of        manufacture of these compounds and polymorphic forms thereof.

I. DEFINITIONS

When used in the context of a chemical group: “hydrogen” means —H;“hydroxy” means —OH; “oxo” means ═O; “carbonyl” means —C(═O)—; “carboxy”means —C(═O)OH (also written as —COOH or —CO₂H); “halo” meansindependently —F, —Cl, —Br or —I; “amino” means —NH₂; “hydroxyamino”means —NHOH; “cyano” means —CN; “isocyanate” means —N═C═O; “azido” means—N₃; in a monovalent context “phosphate” means —OP(O)(OH)₂ or adeprotonated form thereof; in a divalent context “phosphate” means—OP(O)(OH)O— or a deprotonated form thereof; “thio” means ═S; and“sulfonyl” means —S(O)₂—. Any undefined valency on an atom of astructure shown in this application implicitly represents a hydrogenatom bonded to the atom.

The use of the word “a” or “an,” when used in conjunction with the term“comprising” in the claims and/or the specification may mean “one,” butit is also consistent with the meaning of “one or more,” “at least one,”and “one or more than one.”

Throughout this application, the term “about” is used to indicate that avalue includes the inherent variation of error for the device, themethod being employed to determine the value, or the variation thatexists among the study subjects. When used in the context of X-raypowder diffraction, the term “about” is used to indicate a value of ±0.2°2θ from the reported value, preferably a value of ±0.1 °2θ from thereported value. When used in the context of differential scanningcalorimetry or glass transition temperatures, the term “about” is usedto indicate a value of ±10° C. relative to the maximum of the peak,preferably a value of ±2° C. relative to the maximum of the peak. Whenused in another context, the term “about” is used to indicate a value of±10% of the reported value, preferably a value of ±5% of the reportedvalue. It is to be understood that, whenever the term “about” is used, aspecific reference to the exact numerical value indicated is alsoincluded.

The terms “comprise,” “have” and “include” are open-ended linking verbs.Any forms or tenses of one or more of these verbs, such as “comprises,”“comprising,” “has,” “having,” “includes” and “including,” are alsoopen-ended. For example, any method that “comprises,” “has” or“includes” one or more steps is not limited to possessing only those oneor more steps and also covers other unlisted steps.

The term “effective,” as that term is used in the specification and/orclaims, means adequate to accomplish a desired, expected, or intendedresult. “Effective amount,” “Therapeutically effective amount” or“pharmaceutically effective amount” when used in the context of treatinga patient or subject with a compound means that amount of the compoundwhich, when administered to a subject or patient for treating a disease,is sufficient to effect such treatment for the disease.

The term “hydrate” when used as a modifier to a compound means that thecompound has less than one (e.g., hemihydrate), one (e.g., monohydrate),or more than one (e.g., dihydrate) water molecules associated with eachcompound molecule, such as in solid forms of the compound.

As used herein, the term “IC₅₀” refers to an inhibitory dose which is50% of the maximum response obtained. This quantitative measureindicates how much of a particular drug or other substance (inhibitor)is needed to inhibit a given biological, biochemical or chemical process(or component of a process, i.e. an enzyme, cell, cell receptor ormicroorganism) by half.

An “isomer” of a first compound is a separate compound in which eachmolecule contains the same constituent atoms as the first compound, butwhere the configuration of those atoms in three dimensions differs.

As used herein, the term “patient” or “subject” refers to a livingmammalian organism, such as a human, monkey, cow, sheep, goat, dog, cat,mouse, rat, guinea pig, or transgenic species thereof. In certainembodiments, the patient or subject is a non-human mammal. In certainembodiments, the patient or subject is a primate. In certainembodiments, the patient or subject is a human. Non-limiting examples ofhuman subjects are adults, juveniles, infants and fetuses.

As generally used herein “pharmaceutically acceptable” refers to thosecompounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues, organs, and/or bodily fluids of human beings andanimals without excessive toxicity, irritation, allergic response, orother problems or complications commensurate with a reasonablebenefit/risk ratio.

“Pharmaceutically acceptable salts” means salts of compounds of thepresent invention which are pharmaceutically acceptable, as definedabove, and which possess the desired pharmacological activity. Suchsalts include acid addition salts formed with inorganic acids such ashydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,phosphoric acid, and the like; or with organic acids such as1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid,2-naphthalenesulfonic acid, 3-phenylpropionic acid,4,4′-methylenebis(3-hydroxy-2-ene-1-carboxylic acid),4-methylbicyclo[2.2.2]oct-2-ene-1-carboxylic acid, acetic acid,aliphatic mono- and dicarboxylic acids, aliphatic sulfuric acids,aromatic sulfuric acids, benzenesulfonic acid, benzoic acid,camphorsulfonic acid, carbonic acid, cinnamic acid, citric acid,cyclopentanepropionic acid, ethanesulfonic acid, fumaric acid,glucoheptonic acid, gluconic acid, glutamic acid, glycolic acid,heptanoic acid, hexanoic acid, hydroxynaphthoic acid, lactic acid,laurylsulfuric acid, maleic acid, malic acid, malonic acid, mandelicacid, methanesulfonic acid, muconic acid, o-(4-hydroxybenzoyl)benzoicacid, oxalic acid, p-chlorobenzenesulfonic acid, phenyl-substitutedalkanoic acids, propionic acid, p-toluenesulfonic acid, pyruvic acid,salicylic acid, stearic acid, succinic acid, tartaric acid,tertiarybutylacetic acid, trimethylacetic acid, and the like.Pharmaceutically acceptable salts also include base addition salts whichmay be formed when acidic protons present are capable of reacting withinorganic or organic bases. Acceptable inorganic bases include sodiumhydroxide, sodium carbonate, potassium hydroxide, aluminum hydroxide andcalcium hydroxide. Acceptable organic bases include ethanolamine,diethanolamine, triethanolamine, tromethamine, N-methylglucamine and thelike. It should be recognized that the particular anion or cationforming a part of any salt of this invention is not critical, so long asthe salt, as a whole, is pharmacologically acceptable. Additionalexamples of pharmaceutically acceptable salts and their methods ofpreparation and use are presented in Handbook of Pharmaceutical Salts:Properties, and Use (P. H. Stahl & C. G. Wermuth eds., Verlag HelveticaChimica Acta, 2002).

“Prevention” or “preventing” includes: (1) inhibiting the onset of adisease in a subject or patient which may be at risk and/or predisposedto the disease but does not yet experience or display any or all of thepathology or symptomatology of the disease, and/or (2) slowing the onsetof the pathology or symptomatology of a disease in a subject or patientwhich may be at risk and/or predisposed to the disease but does not yetexperience or display any or all of the pathology or symptomatology ofthe disease. “Prodrug” means a compound that is convertible in vivometabolically into an inhibitor according to the present invention. Theprodrug itself may or may not also have activity with respect to a giventarget protein. For example, a compound comprising a hydroxy group maybe administered as an ester that is converted by hydrolysis in vivo tothe hydroxy compound. Suitable esters that may be converted in vivo intohydroxy compounds include acetates, citrates, lactates, phosphates,tartrates, malonates, oxalates, salicylates, propionates, succinates,fumarates, maleates, methylene-bis-β-hydroxynaphthoate, gentisates,isethionates, di-p-toluoyltartrates, methanesulfonates,ethanesulfonates, benzenesulfonates, p-toluenesulfonates,cyclohexylsulfamates, quinates, esters of amino acids, and the like.Similarly, a compound comprising an amine group may be administered asan amide that is converted by hydrolysis in vivo to the amine compound.

A “stereoisomer” or “optical isomer” is an isomer of a given compound inwhich the same atoms are bonded to the same other atoms, but where theconfiguration of those atoms in three dimensions differs. “Enantiomers”are stereoisomers of a given compound that are mirror images of eachother, like left and right hands. “Diastereomers” are stereoisomers of agiven compound that are not enantiomers. Chiral molecules contain achiral center, also referred to as a stereocenter or stereogenic center,which is any point, though not necessarily an atom, in a moleculebearing groups such that an interchanging of any two groups leads to astereoisomer. In organic compounds, the chiral center is typically acarbon, phosphorus or sulfur atom, though it is also possible for otheratoms to be stereocenters in organic and inorganic compounds. A moleculecan have multiple stereocenters, giving it many stereoisomers. Incompounds whose stereoisomerism is due to tetrahedral stereogeniccenters (e.g., tetrahedral carbon), the total number of hypotheticallypossible stereoisomers will not exceed 2n, where n is the number oftetrahedral stereocenters. Molecules with symmetry frequently have fewerthan the maximum possible number of stereoisomers. A 50:50 mixture ofenantiomers is referred to as a racemic mixture. Alternatively, amixture of enantiomers can be enantiomerically enriched so that oneenantiomer is present in an amount greater than 50%. Typically,enantiomers and/or diastereomers can be resolved or separated usingtechniques known in the art. It is contemplated that for anystereocenter or axis of chirality for which stereochemistry has not beendefined, that stereocenter or axis of chirality can be present in its Rform, S form, or as a mixture of the R and S forms, including racemicand non-racemic mixtures. As used herein, the phrase “substantially freefrom other stereoisomers” means that the composition contains ≤15%, morepreferably ≤10%, even more preferably ≤5%, or most preferably ≤1% ofanother stereoisomer(s).

“Treatment” or “treating” includes (1) inhibiting a disease in a subjector patient experiencing or displaying the pathology or symptomatology ofthe disease (e.g., arresting further development of the pathology and/orsymptomatology), (2) ameliorating a disease in a subject or patient thatis experiencing or displaying the pathology or symptomatology of thedisease (e.g., reversing the pathology and/or symptomatology), and/or(3) effecting any measurable decrease in a disease in a subject orpatient that is experiencing or displaying the pathology orsymptomatology of the disease.

In the context of this disclosure, the formulas:

represent the same structures. When a dot is drawn on a carbon, the dotindicates that the hydrogen atom attached to that carbon is coming outof the plane of the page.

The above definitions supersede any conflicting definition in any of thereferences that are incorporated by reference herein. The fact thatcertain terms are defined, however, should not be considered asindicative that any term that is undefined is indefinite. Rather, allterms used are believed to describe the invention in terms such that oneof ordinary skill can appreciate the scope and practice the presentinvention.

II. RTA 408 AND SYNTHETIC METHODS

RTA 408 can be prepared according to the methods described in thesection below. These methods can be further modified and optimized usingthe principles and techniques of organic chemistry as applied by aperson skilled in the art. Such principles and techniques are taught,for example, in March's Advanced Organic Chemistry: Reactions,Mechanisms, and Structure (2007), which is incorporated by referenceherein.

It should be recognized that the particular anion or cation forming apart of any salt of this invention is not critical, so long as the salt,as a whole, is pharmacologically acceptable. Additional examples ofpharmaceutically acceptable salts and their methods of preparation anduse are presented in Handbook of Pharmaceutical Salts: Properties, andUse (2002), which is incorporated herein by reference.

RTA 408 may also exist in prodrug form. Since prodrugs are known toenhance numerous desirable qualities of pharmaceuticals, e.g.,solubility, bioavailability, manufacturing, etc., the compounds employedin some methods of the invention may, if desired, be delivered inprodrug form. Thus, the invention contemplates prodrugs of compounds ofthe present invention as well as methods of delivering prodrugs.Prodrugs of the compounds employed in the invention may be prepared bymodifying functional groups present in the compound in such a way thatthe modifications are cleaved, either in routine manipulation or invivo, to the parent compound. Accordingly, prodrugs include, forexample, compounds described herein in which a hydroxy, amino, orcarboxy group is bonded to any group that, when the prodrug isadministered to a patient, cleaves to form a hydroxy, amino, orcarboxylic acid, respectively.

RTA 408 may contain one or more asymmetrically-substituted carbon ornitrogen atoms, and may be isolated in optically active or racemic form.Thus, all chiral, diastereomeric, racemic form, epimeric form, and allgeometric isomeric forms of a structure are intended, unless thespecific stereochemistry or isomeric form is specifically indicated. RTA408 may occur as racemates and racemic mixtures, single enantiomers,diastereomeric mixtures and individual diastereomers. In someembodiments, a single diastereomer is obtained. The chiral centers ofRTA 408 according to the present invention can have the S or the Rconfiguration.

In addition, atoms making up RTA 408 of the present invention areintended to include all isotopic forms of such atoms. Isotopes, as usedherein, include those atoms having the same atomic number but differentmass numbers. By way of general example and without limitation, isotopesof hydrogen include tritium and deuterium, and isotopes of carboninclude ¹³C and ¹⁴C. Similarly, it is contemplated that one or morecarbon atom(s) of a compound of the present invention may be replaced bya silicon atom(s). Furthermore, it is contemplated that one or moreoxygen atom(s) of RTA 408 may be replaced by a sulfur or seleniumatom(s).

RTA 408 and polymorphic form thereof may also have the advantage thatthey may be more efficacious than, be less toxic than, be longer actingthan, be more potent than, produce fewer side effects than, be moreeasily absorbed than, and/or have a better pharmacokinetic profile(e.g., higher oral bioavailability and/or lower clearance) than, and/orhave other useful pharmacological, physical, or chemical advantagesover, compounds known in the prior art for use in the indications statedherein.

III. POLYMORPHIC FORMS OF RTA 408

In some embodiments, the present invention provides different solidforms of RTA 408, including solvates thereof. A polymorphism study wasperformed, and RTA 408 was found in two, essentially solvent-free,crystalline forms (Form A and Form B). For a description of the classes,see Table 1 below. Crystalline Form A is metastable and has a meltingpoint at 181.98° C. and ΔH fusion=42.01 J/g. This form may have utilityfor obtaining amorphous forms of RTA 408 or in extrusion formulations.Crystalline Form A may be slightly hygroscopic (mass loss of ˜0.5 wt. %in TGA-MS, FIG. 55). Crystalline Form B has greater thermodynamicstability than Form A as indicated by a higher melting point (250.10°C.) and greater enthalpy of fusion (ΔH fusion=47.85 J/g). Greaterchemical and physical stability is expected for Form B compared to FormA both at ambient and elevated temperatures. A minimal amount of surfacewater may exist on Form B as indicated by TGA-MS (FIG. 58).

The new forms were characterized by PXRD (Table 8 and Table 9).

TABLE 1 Summary of Solid Forms Form Melting Point Enthalpy of Fusion A181.98° C. 42.01 J/g B 250.10° C. 47.85 J/g

IV. DISEASES ASSOCIATED WITH INFLAMMATION AND/OR OXIDATIVE STRESS

Inflammation is a biological process that provides resistance toinfectious or parasitic organisms and the repair of damaged tissue.Inflammation is commonly characterized by localized vasodilation,redness, swelling, and pain, the recruitment of leukocytes to the siteof infection or injury, production of inflammatory cytokines, such asTNF-α and IL-1, and production of reactive oxygen or nitrogen species,such as hydrogen peroxide, superoxide, and peroxynitrite. In laterstages of inflammation, tissue remodeling, angiogenesis, and scarformation (fibrosis) may occur as part of the wound healing process.Under normal circumstances, the inflammatory response is regulated,temporary, and is resolved in an orchestrated fashion once the infectionor injury has been dealt with adequately. However, acute inflammationcan become excessive and life-threatening if regulatory mechanisms fail.Alternatively, inflammation can become chronic and cause cumulativetissue damage or systemic complications. Based at least on the evidencepresented herein, RTA 408 can be used in the treatment or prevention ofinflammation or diseases associated with inflammation.

Many serious and intractable human diseases involve dysregulation ofinflammatory processes, including diseases such as cancer,atherosclerosis, and diabetes, which were not traditionally viewed asinflammatory conditions. In the case of cancer, the inflammatoryprocesses are associated with tumor formation, progression, metastasis,and resistance to therapy. Atherosclerosis, long viewed as a disorder oflipid metabolism, is now understood to be primarily an inflammatorycondition, with activated macrophages playing an important role in theformation and eventual rupture of atherosclerotic plaques. Activation ofinflammatory signaling pathways has also been shown to play a role inthe development of insulin resistance, as well as in the peripheraltissue damage associated with diabetic hyperglycemia. Excessiveproduction of reactive oxygen species and reactive nitrogen species,such as superoxide, hydrogen peroxide, nitric oxide, and peroxynitrite,is a hallmark of inflammatory conditions. Evidence of dysregulatedperoxynitrite production has been reported in a wide variety of diseases(Szabo et al., 2007; Schulz et al., 2008; Forstermann, 2006; Pall,2007).

Autoimmune diseases such as rheumatoid arthritis, lupus, psoriasis, andmultiple sclerosis involve inappropriate and chronic activation ofinflammatory processes in affected tissues, arising from dysfunction ofself vs. non-self recognition and response mechanisms in the immunesystem. In neurodegenerative diseases such as Alzheimer's andParkinson's diseases, neural damage is correlated with activation ofmicroglia and elevated levels of pro-inflammatory proteins, such asinducible nitric oxide synthase (iNOS). Chronic organ failure, such asrenal failure, heart failure, liver failure, and chronic obstructivepulmonary disease, is closely associated with the presence of chronicoxidative stress and inflammation, leading to the development offibrosis and eventual loss of organ function. Oxidative stress invascular endothelial cells, which line major and minor blood vessels,can lead to endothelial dysfunction and is believed to be an importantcontributing factor in the development of systemic cardiovasculardisease, complications of diabetes, chronic kidney disease and otherforms of organ failure, and a number of other aging-related diseases,including degenerative diseases of the central nervous system and theretina.

Many other disorders involve oxidative stress and inflammation inaffected tissues, including inflammatory bowel disease; inflammatoryskin diseases; mucositis and dermatitis related to radiation therapy andchemotherapy; eye diseases, such as uveitis, glaucoma, maculardegeneration, and various forms of retinopathy; transplant failure andrejection; ischemia-reperfusion injury; chronic pain; degenerativeconditions of the bones and joints, including osteoarthritis andosteoporosis; asthma and cystic fibrosis; seizure disorders; andneuropsychiatric conditions, including schizophrenia, depression,bipolar disorder, post-traumatic stress disorder, attention deficitdisorders, autism-spectrum disorders, and eating disorders, such asanorexia nervosa. Dysregulation of inflammatory signaling pathways isbelieved to be a major factor in the pathology of muscle wastingdiseases, including muscular dystrophy and various forms of cachexia.

A variety of life-threatening acute disorders also involve dysregulatedinflammatory signaling, including acute organ failure involving thepancreas, kidneys, liver, or lungs, myocardial infarction or acutecoronary syndrome, stroke, septic shock, trauma, severe burns, andanaphylaxis.

Many complications of infectious diseases also involve dysregulation ofinflammatory responses. Although an inflammatory response can killinvading pathogens, an excessive inflammatory response can also be quitedestructive and in some cases can be a primary source of damage ininfected tissues. Furthermore, an excessive inflammatory response canalso lead to systemic complications due to overproduction ofinflammatory cytokines, such as TNF-α and IL-1. This is believed to be afactor in mortality arising from severe influenza, severe acuterespiratory syndrome, and sepsis.

The aberrant or excessive expression of either iNOS or cyclooxygenase-2(COX-2) has been implicated in the pathogenesis of many diseaseprocesses. For example, it is clear that NO is a potent mutagen (Tamirand Tannebaum, 1996), and that nitric oxide can also activate COX-2(Salvemini et al., 1994). Furthermore, there is a marked increase iniNOS in rat colon tumors induced by the carcinogen, azoxymethane(Takahashi et al., 1997). A series of synthetic triterpenoid analogs ofoleanolic acid have been shown to be powerful inhibitors of cellularinflammatory processes, such as the induction by IFN-γ of induciblenitric oxide synthase (iNOS) and of COX-2 in mouse macrophages. SeeHonda et al. (2000a), Honda et al. (2000b), and Honda et al. (2002),which are all incorporated herein by reference.

In one aspect, RTA 408 disclosed herein is in part characterized by itsability to inhibit the production of nitric oxide in macrophage-derivedRAW 264.7 cells induced by exposure to γ-interferon. RTA 408 is furthercharacterized by the ability to induce the expression of antioxidantproteins, such as NQO1, and reduce the expression of pro-inflammatoryproteins, such as COX-2 and inducible nitric oxide synthase (iNOS).These properties are relevant to the treatment of a wide array ofdiseases and disorders involving oxidative stress and dysregulation ofinflammatory processes, including cancer, complications from localizedor total-body exposure to ionizing radiation, mucositis and dermatitisresulting from radiation therapy or chemotherapy, autoimmune diseases,cardiovascular diseases, including atherosclerosis, ischemia-reperfusioninjury, acute and chronic organ failure, including renal failure andheart failure, respiratory diseases, diabetes and complications ofdiabetes, severe allergies, transplant rejection, graft-versus-hostdisease, neurodegenerative diseases, diseases of the eye and retina,acute and chronic pain, degenerative bone diseases, includingosteoarthritis and osteoporosis, inflammatory bowel diseases, dermatitisand other skin diseases, sepsis, burns, seizure disorders, andneuropsychiatric disorders.

In another aspect, RTA 408 may be used for treating a subject having acondition such as eye diseases. For example, uveitis, maculardegeneration (both the dry form and wet form), glaucoma, diabeticmacular edema, blepharitis, diabetic retinopathy, diseases and disordersof the corneal endothelium such as Fuchs endothelial corneal dystrophy,post-surgical inflammation, dry eye, allergic conjunctivitis and otherforms of conjunctivitis are non-limiting examples of eye diseases thatcould be treated with RTA 408.

In another aspect, RTA 408 may be used for treating a subject having acondition such as skin diseases or disorders. For example, dermatitis,including allergic dermatitis, atopic dermatitis, dermatitis due tochemical exposure, and radiation-induced dermatitis; thermal or chemicalburns; chronic wounds including diabetic ulcers, pressure sores, andvenous ulcers; acne; alopecia including baldness and drug-inducedalopecia; other disorders of the hair follicle; epidermolysis bullosa;sunburn and its complications; disorders of skin pigmentation includingvitiligo; aging-related skin conditions; post-surgical wound healing;prevention or reduction of scarring from skin injury, surgery, or burns;psoriasis; dermatological manifestations of autoimmune diseases orgraft-versus host disease; prevention or treatment of skin cancer;disorders involving hyperproliferation of skin cells such ashyperkeratosis is a non-limiting example of skin diseases that could betreated with RTA 408.

Without being bound by theory, the activation of theantioxidant/anti-inflammatory Keap1/Nrf2/ARE pathway is believed to beimplicated in both the anti-inflammatory and anti-carcinogenicproperties of the compound disclosed herein.

In another aspect, RTA 408 may be used for treating a subject having acondition caused by elevated levels of oxidative stress in one or moretissues. Oxidative stress results from abnormally high or prolongedlevels of reactive oxygen species, such as superoxide, hydrogenperoxide, nitric oxide, and peroxynitrite (formed by the reaction ofnitric oxide and superoxide). The oxidative stress may be accompanied byeither acute or chronic inflammation. The oxidative stress may be causedby mitochondrial dysfunction, by activation of immune cells, such asmacrophages and neutrophils, by acute exposure to an external agent,such as ionizing radiation or a cytotoxic chemotherapy agent (e.g.,doxorubicin), by trauma or other acute tissue injury, byischemia/reperfusion, by poor circulation or anemia, by localized orsystemic hypoxia or hyperoxia, by elevated levels of inflammatorycytokines and other inflammation-related proteins, and/or by otherabnormal physiological states, such as hyperglycemia or hypoglycemia.

In animal models of many such conditions, stimulating expression ofinducible heme oxygenase (HO-1), a target gene of the Nrf2 pathway, hasbeen shown to have a significant therapeutic effect including in modelsof myocardial infarction, renal failure, transplant failure andrejection, stroke, cardiovascular disease, and autoimmune disease (e.g.,Sacerdoti et al., 2005; Abraham & Kappas, 2005; Bach, 2006; Araujo etal., 2003; Liu et al., 2006; Ishikawa et al., 2001; Kruger et al., 2006;Satoh et al., 2006; Zhou et al., 2005; Morse and Choi, 2005; Morse andChoi, 2002). This enzyme breaks free heme down into iron, carbonmonoxide (CO), and biliverdin (which is subsequently converted to thepotent antioxidant molecule, bilirubin).

In another aspect, RTA 408 may be used in preventing or treating tissuedamage or organ failure, acute and chronic, resulting from oxidativestress exacerbated by inflammation. Examples of diseases that fall inthis category include heart failure, liver failure, transplant failureand rejection, renal failure, pancreatitis, fibrotic lung diseases(cystic fibrosis, COPD, and idiopathic pulmonary fibrosis, amongothers), diabetes (including complications), atherosclerosis,ischemia-reperfusion injury, glaucoma, stroke, autoimmune disease,autism, macular degeneration, and muscular dystrophy. For example, inthe case of autism, studies suggest that increased oxidative stress inthe central nervous system may contribute to the development of thedisease (Chauhan and Chauhan, 2006).

Evidence also links oxidative stress and inflammation to the developmentand pathology of many other disorders of the central nervous system,including psychiatric disorders, such as psychosis, major depression,and bipolar disorder; seizure disorders, such as epilepsy; pain andsensory syndromes, such as migraine, neuropathic pain, or tinnitus; andbehavioral syndromes, such as the attention deficit disorders. See,e.g., Dickerson et al., 2007; Hanson et al., 2005; Kendall-Tackett,2007; Lencz et al., 2007; Dudhgaonkar et al., 2006; Lee et al., 2007;Morris et al., 2002; Ruster et al., 2005; McIver et al., 2005;Sarchielli et al., 2006; Kawakami et al., 2006; Ross et al., 2003, whichare all incorporated by reference herein. For example, elevated levelsof inflammatory cytokines, including TNF, interferon-γ, and IL-6, areassociated with major mental illness (Dickerson et al., 2007).Microglial activation has also been linked to major mental illness.Therefore, downregulating inflammatory cytokines and inhibitingexcessive activation of microglia could be beneficial in patients withschizophrenia, major depression, bipolar disorder, autism-spectrumdisorders, and other neuropsychiatric disorders.

Accordingly, in pathologies involving oxidative stress alone oroxidative stress exacerbated by inflammation, treatment may compriseadministering to a subject a therapeutically effective amount of acompound of this invention, such as those described above or throughoutthis specification. Treatment may be administered preventively, inadvance of a predictable state of oxidative stress (e.g., organtransplantation or the administration of radiation therapy to a cancerpatient), or it may be administered therapeutically in settingsinvolving established oxidative stress and inflammation. In someinstances, such as a cancer patient receiving radiation therapy orchemotherapy (or both), the compound of the invention may beadministered both before and after the radiation or chemotherapy, or maybe administered in combination with the other therapies. Depending onthe nature of the radiation therapy or chemotherapy, variouscombinations of pre-treatment, post-treatment, or simultaneousadministration of the compound of the invention may be used. Thecompound of the invention may prevent or reduce the severity of sideeffects associated with the radiation therapy or chemotherapy. Becausesuch side effects may be dose-limiting, their reduction or preventionmay allow higher or more frequent dosing of the radiation therapy orchemotherapy, resulting in greater efficacy. Alternatively, as shownherein, use of the compound of the invention in combination with theradiation therapy or chemotherapy may enhance the efficacy of a givendose of radiation or chemotherapy. In part, this combinatorial efficacymay result from inhibition of the activity of the pro-inflammatorytranscription factor NF-κB by the compound of the invention. NF-κB isoften chronically activated in cancer cells, and such activation isassociated with resistance to therapy and promotion of tumor progression(e.g., Karin M, Nature. 2006 May 25; 441(7092):431-6; Aghajan et al., JGastroenterol Hepatol. 2012 March; 27 Suppl 2:10-4). Other transcriptionfactors that promote inflammation and cancer, such as STAT3 (e.g., He Gand Karin M, Cell Res. 2011 January; 21(1):159-68; Grivennikov S I andKarin M, Cytokine Growth Factor Rev. 2010 February; 21(1):11-9), mayalso be inhibited by the compound of the invention.

RTA 408 may be used to treat or prevent inflammatory conditions, such assepsis, dermatitis, autoimmune disease, and osteoarthritis. RTA 408 mayalso be used to treat or prevent inflammatory pain and/or neuropathicpain, for example, by inducing Nrf2 and/or inhibiting NF-κB.

RTA 408 may also be used to treat or prevent diseases, such as cancer,inflammation, Alzheimer's disease, Parkinson's disease, multiplesclerosis, autism, amyotrophic lateral sclerosis, Huntington's disease,autoimmune diseases, such as rheumatoid arthritis, lupus, Crohn'sdisease, and psoriasis, inflammatory bowel disease, all other diseaseswhose pathogenesis is believed to involve excessive production of eithernitric oxide or prostaglandins, and pathologies involving oxidativestress alone or oxidative stress exacerbated by inflammation.

Another aspect of inflammation is the production of inflammatoryprostaglandins, such as prostaglandin E. RTA 408 may be used to promotevasodilation, plasma extravasation, localized pain, elevatedtemperature, and other symptoms of inflammation. The inducible form ofthe enzyme COX-2 is associated with their production, and high levels ofCOX-2 are found in inflamed tissues. Consequently, inhibition of COX-2may relieve many symptoms of inflammation and a number of importantanti-inflammatory drugs (e.g., ibuprofen and celecoxib) act byinhibiting COX-2 activity. It has been demonstrated that a class ofcyclopentenone prostaglandins (cyPGs) (e.g., 15-deoxy prostaglandin J2,a.k.a. PGJ2) plays a role in stimulating the orchestrated resolution ofinflammation (e.g., Rajakariar et al., 2007). COX-2 is also associatedwith the production of cyclopentenone prostaglandins. Consequently,inhibition of COX-2 may interfere with the full resolution ofinflammation, potentially promoting the persistence of activated immunecells in tissues and leading to chronic, “smoldering” inflammation. Thiseffect may be responsible for the increased incidence of cardiovasculardisease in patients using selective COX-2 inhibitors for long periods oftime.

In one aspect, RTA 408 may be used to control the production ofpro-inflammatory cytokines within the cell by selectively activatingregulatory cysteine residues (RCRs) on proteins that regulate theactivity of redox-sensitive transcription factors. Activation of RCRs bycyPGs has been shown to initiate a pro-resolution program in which theactivity of the antioxidant and cytoprotective transcription factor Nrf2is potently induced and the activities of the pro-oxidant andpro-inflammatory transcription factors NF-κB and the STATs aresuppressed. In some embodiments, RTA 408 may be used to increase theproduction of antioxidant and reductive molecules (NQO1, HO-1, SOD1,γ-GCS) and decrease oxidative stress and the production of pro-oxidantand pro-inflammatory molecules (iNOS, COX-2, TNF-α). In someembodiments, RTA 408 may be used to cause the cells that host theinflammatory event to revert to a non-inflammatory state by promotingthe resolution of inflammation and limiting excessive tissue damage tothe host.

A. Cancer

Further, RTA 408 may be used to induce apoptosis in tumor cells, toinduce cell differentiation, to inhibit cancer cell proliferation, toinhibit an inflammatory response, and/or to function in achemopreventative capacity. For example, RTA 408 has one or more of thefollowing properties: (1) an ability to induce apoptosis anddifferentiate both malignant and non-malignant cells, (2) an activity atsub-micromolar or nanomolar levels as an inhibitor of proliferation ofmany malignant or premalignant cells, (3) an ability to suppress the denovo synthesis of the inflammatory enzyme inducible nitric oxidesynthase (iNOS), (4) an ability to inhibit NF-κB activation, and (5) anability to induce the expression of heme oxygenase-1 (HO-1).

The levels of iNOS and COX-2 are elevated in certain cancers and havebeen implicated in carcinogenesis and COX-2 inhibitors have been shownto reduce the incidence of primary colonic adenomas in humans (Rostom etal., 2007; Brown and DuBois, 2005; Crowel et al., 2003). iNOS isexpressed in myeloid-derived suppressor cells (MDSCs) (Angulo et al.,2000) and COX-2 activity in cancer cells has been shown to result in theproduction of prostaglandin E2 (PGE2), which has been shown to inducethe expression of arginase in MDSCs (Sinha et al., 2007). Arginase andiNOS are enzymes that utilize L-arginine as a substrate and produceL-ornithine and urea, and L-citrulline and NO, respectively. Thedepletion of arginine from the tumor microenvironment by MDSCs, combinedwith the production of NO and peroxynitrite has been shown to inhibitproliferation and induce apoptosis of T cells (Bronte et al., 2003).Inhibition of COX-2 and iNOS has been shown to reduce the accumulationof MDSCs, restore cytotoxic activity of tumor-associated T cells, anddelay tumor growth (Sinha et al., 2007; Mazzoni et al., 2002; Zhou etal., 2007).

Inhibition of the NF-κB and JAK/STAT signaling pathways has beenimplicated as a strategy to inhibit proliferation of cancer epithelialcells and induce their apoptosis. Activation of STAT3 and NF-κB has beenshown to result in suppression of apoptosis in cancer cells, andpromotion of proliferation, invasion, and metastasis. Many of the targetgenes involved in these processes have been shown to betranscriptionally regulated by both NF-κB and STAT3 (Yu et al., 2007).

In addition to their direct roles in cancer epithelial cells, NF-κB andSTAT3 also have important roles in other cells found within the tumormicroenvironment. Experiments in animal models have demonstrated thatNF-κB is required in both cancer cells and hematopoeitic cells topropagate the effects of inflammation on cancer initiation andprogression (Greten et al., 2004). NF-κB inhibition in cancer andmyeloid cells reduces the number and size, respectively, of theresultant tumors. Activation of STAT3 in cancer cells results in theproduction of several cytokines (IL-6, IL-10) which suppress thematuration of tumor-associated dendritic cells (DC). Furthermore, STAT3is activated by these cytokines in the dendritic cells themselves.Inhibition of STAT3 in mouse models of cancer restores DC maturation,promotes antitumor immunity, and inhibits tumor growth (Kortylewski etal., 2005).

B. Treatment of Multiple Sclerosis and Other NeurodegenerativeConditions

The compound and methods of this invention may be used for treatingpatients for multiple sclerosis (MS). MS is known to be an inflammatorycondition of the central nervous system (Williams et al., 1994; Merrilland Benvenist, 1996; Genain and Nauser, 1997). Based on severalinvestigations, there is evidence suggesting that inflammatory,oxidative, and/or immune mechanisms are involved in the pathogenesis ofAlzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateralsclerosis (ALS), and MS (Bagasra et al., 1995; McGeer and McGeer, 1995;Simonian and Coyle, 1996; Kaltschmidt et al., 1997). Both reactiveastrocytes and activated microglia have been implicated in causation ofneurodegenerative disease (NDD) and neuroinflammatory disease (NID);there has been a particular emphasis on microglia as cells thatsynthesize both NO and prostaglandins as products of the respectiveenzymes, iNOS and COX-2. De novo formation of these enzymes may bedriven by inflammatory cytokines such as interferon-γ or interleukin-1.In turn, excessive production of NO may lead to inflammatory cascadesand/or oxidative damage in cells and tissues of many organs, includingneurons and oligodendrocytes of the nervous system, with consequentmanifestations in AD and MS, and possible PD and ALS (Coyle andPuttfarcken, 1993; Beal, 1996; Merrill and Benvenist, 1996; Simonian andCoyle, 1996; Vodovotz et al., 1996). Epidemiologic data indicate thatchronic use of NSAID's which block synthesis of prostaglandins fromarachidonate, markedly lower the risk for development of AD (McGeer etal., 1996; Stewart et al., 1997). Thus, agents that block formation ofNO and prostaglandins, may be used in approaches to prevention andtreatment of NDD. Successful therapeutic candidates for treating such adisease typically require an ability to penetrate the blood-brainbarrier. See, for example, U.S. Patent Publication 2009/0060873, whichis incorporated by reference herein in its entirety.

C. Neuroinflammation

The compound and methods of this invention may be used for treatingpatients with neuroinflammation. Neuroinflammation encapsulates the ideathat microglial and astrocytic responses and actions in the centralnervous system have a fundamentally inflammation-like character, andthat these responses are central to the pathogenesis and progression ofa wide variety of neurological disorders. This idea originated in thefield of Alzheimer's disease (Griffin et al., 1989; Rogers et al.,1988), where it has revolutionized our understanding of this disease(Akiyama et al., 2000). These ideas have been extended to otherneurodegenerative diseases (Eikelenboom et al., 2002; Ishizawa andDickson, 2001), to ischemic/toxic diseases (Gehrmann et al., 1995;Touzani et al., 1999), to tumor biology (Graeber et al., 2002) and evento normal brain development.

Neuroinflammation incorporates a wide spectrum of complex cellularresponses that include activation of microglia and astrocytes andinduction of cytokines, chemokines, complement proteins, acute phaseproteins, oxidative injury, and related molecular processes. Theseevents may have detrimental effects on neuronal function, leading toneuronal injury, further glial activation, and ultimatelyneurodegeneration.

D. Treatment of Renal Failure

The compound and methods of this invention may be used for treatingpatients with renal failure. See U.S. patent application Ser. No.12/352,473, which is incorporated by reference herein in its entirety.Another aspect of the present disclosure concerns new methods andcompounds for the treatment and prevention of renal disease. Renalfailure, resulting in inadequate clearance of metabolic waste productsfrom the blood and abnormal concentrations of electrolytes in the blood,is a significant medical problem throughout the world, especially indeveloped countries. Diabetes and hypertension are among the mostimportant causes of chronic renal failure, also known as chronic kidneydisease (CKD), but it is also associated with other conditions such aslupus. Acute renal failure may arise from exposure to certain drugs(e.g., acetaminophen) or toxic chemicals, or from ischemia-reperfusioninjury associated with shock or surgical procedures such astransplantation, and may result in chronic renal failure. In manypatients, renal failure advances to a stage in which the patientrequires regular dialysis or kidney transplantation to continue living.Both of these procedures are highly invasive and associated withsignificant side effects and quality of life issues. Although there areeffective treatments for some complications of renal failure, such ashyperparathyroidism and hyperphosphatemia, no available treatment hasbeen shown to halt or reverse the underlying progression of renalfailure. Thus, agents that can improve compromised renal function wouldrepresent a significant advance in the treatment of renal failure.

Inflammation contributes significantly to the pathology of CKD. There isalso a strong mechanistic link between oxidative stress and renaldysfunction. The NF-κB signaling pathway plays an important role in theprogression of CKD as NF-κB regulates the transcription of MCP-1, achemokine that is responsible for the recruitment ofmonocytes/macrophages resulting in an inflammatory response thatultimately injures the kidney (Wardle, 2001). The Keap1/Nrf2/ARE pathwaycontrols the transcription of several genes encoding antioxidantenzymes, including heme oxygenase-1 (HO-1). Ablation of the Nrf2 gene infemale mice results in the development of lupus-like glomerularnephritis (Yoh et al., 2001). Furthermore, several studies havedemonstrated that HO-1 expression is induced in response to renal damageand inflammation and that this enzyme and its products—bilirubin andcarbon monoxide—play a protective role in the kidney (Nath et al.,2006).

The glomerulus and the surrounding Bowman's capsule constitute the basicfunctional unit of the kidney. Glomerular filtration rate (GFR) is thestandard measure of renal function. Creatinine clearance is commonlyused to measure GFR. However, the level of serum creatinine is commonlyused as a surrogate measure of creatinine clearance. For instance,excessive levels of serum creatinine are generally accepted to indicateinadequate renal function and reductions in serum creatinine over timeare accepted as an indication of improved renal function. Normal levelsof creatinine in the blood are approximately 0.6 to 1.2 milligrams (mg)per deciliter (dl) in adult males and 0.5 to 1.1 milligrams perdeciliter in adult females.

Acute kidney injury (AKI) can occur following ischemia-reperfusion,treatment with certain pharmacological agents such as cisplatin andrapamycin, and intravenous injection of radiocontrast media used inmedical imaging. As in CKD, inflammation and oxidative stress contributeto the pathology of AKI. The molecular mechanisms underlyingradiocontrast-induced nephropathy (RCN) are not well understood;however, it is likely that a combination of events including prolongedvasoconstriction, impaired kidney autoregulation, and direct toxicity ofthe contrast media all contribute to renal failure (Tumlin et al.,2006). Vasoconstriction results in decreased renal blood flow and causesischemia-reperfusion and the production of reactive oxygen species. HO-1is strongly induced under these conditions and has been demonstrated toprevent ischemia-reperfusion injury in several different organs,including the kidney (Nath et al., 2006). Specifically, induction ofHO-1 has been shown to be protective in a rat model of RCN (Goodman etal., 2007). Reperfusion also induces an inflammatory response, in partthough activation of NF-κB signaling (Nichols, 2004). Targeting NF-κBhas been proposed as a therapeutic strategy to prevent organ damage(Zingarelli et al., 2003).

E. Cardiovascular Disease

The compound and methods of this invention may be used for treatingpatients with cardiovascular disease. See U.S. patent application Ser.No. 12/352,473, which is incorporated by reference herein in itsentirety. Cardiovascular (CV) disease is among the most important causesof mortality worldwide, and is the leading cause of death in manydeveloped nations. The etiology of CV disease is complex, but themajority of causes are related to inadequate or completely disruptedsupply of blood to a critical organ or tissue. Frequently such acondition arises from the rupture of one or more atheroscleroticplaques, which leads to the formation of a thrombus that blocks bloodflow in a critical vessel. Such thrombosis is the principal cause ofheart attacks, in which one or more of the coronary arteries is blockedand blood flow to the heart itself is disrupted. The resulting ischemiais highly damaging to cardiac tissue, both from lack of oxygen duringthe ischemic event and from excessive formation of free radicals afterblood flow is restored (a phenomenon known as ischemia-reperfusioninjury). Similar damage occurs in the brain during a thrombotic stroke,when a cerebral artery or other major vessel is blocked by thrombosis.Hemorrhagic strokes, in contrast, involve rupture of a blood vessel andbleeding into the surrounding brain tissue. This creates oxidativestress in the immediate area of the hemorrhage, due to the presence oflarge amounts of free heme and other reactive species, and ischemia inother parts of the brain due to compromised blood flow. Subarachnoidhemorrhage, which is frequently accompanied by cerebral vasospasm, alsocauses ischemia/reperfusion injury in the brain.

Alternatively, atherosclerosis may be so extensive in critical bloodvessels that stenosis (narrowing of the arteries) develops and bloodflow to critical organs (including the heart) is chronicallyinsufficient. Such chronic ischemia can lead to end-organ damage of manykinds, including the cardiac hypertrophy associated with congestiveheart failure.

Atherosclerosis, the underlying defect leading to many forms ofcardiovascular disease, occurs when a physical defect or injury to thelining (endothelium) of an artery triggers an inflammatory responseinvolving the proliferation of vascular smooth muscle cells and theinfiltration of leukocytes into the affected area. Ultimately, acomplicated lesion known as an atherosclerotic plaque may form, composedof the above-mentioned cells combined with deposits ofcholesterol-bearing lipoproteins and other materials (e.g., Hansson etal., 2006).

Pharmaceutical treatments for cardiovascular disease include preventivetreatments, such as the use of drugs intended to lower blood pressure orcirculating levels of cholesterol and lipoproteins, as well astreatments designed to reduce the adherent tendencies of platelets andother blood cells (thereby reducing the rate of plaque progression andthe risk of thrombus formation). More recently, drugs such asstreptokinase and tissue plasminogen activator have been introduced andare used to dissolve the thrombus and restore blood flow. Surgicaltreatments include coronary artery bypass grafting to create analternative blood supply, balloon angioplasty to compress plaque tissueand increase the diameter of the arterial lumen, and carotidendarterectomy to remove plaque tissue in the carotid artery. Suchtreatments, especially balloon angioplasty, may be accompanied by theuse of stents, expandable mesh tubes designed to support the arterywalls in the affected area and keep the vessel open. Recently, the useof drug-eluting stents has become common in order to preventpost-surgical restenosis (renarrowing of the artery) in the affectedarea. These devices are wire stents coated with a biocompatible polymermatrix containing a drug that inhibits cell proliferation (e.g.,paclitaxel or rapamycin). The polymer allows a slow, localized releaseof the drug in the affected area with minimal exposure of non-targettissues. Despite the significant benefits offered by such treatments,mortality from cardiovascular disease remains high and significant unmetneeds in the treatment of cardiovascular disease remain.

As noted above, induction of HO-1 has been shown to be beneficial in avariety of models of cardiovascular disease, and low levels of HO-1expression have been clinically correlated with elevated risk of CVdisease. Compounds of the invention, therefore, may be used in treatingor preventing a variety of cardiovascular disorders including but notlimited to atherosclerosis, hypertension, myocardial infarction, chronicheart failure, stroke, subarachnoid hemorrhage, and restenosis.

F. Diabetes

The compound and methods of this invention may be used for treatingpatients with diabetes. See U.S. patent application Ser. No. 12/352,473,which is incorporated by reference herein in its entirety. Diabetes is acomplex disease characterized by the body's failure to regulatecirculating levels of glucose. This failure may result from a lack ofinsulin, a peptide hormone that regulates both the production andabsorption of glucose in various tissues. Deficient insulin compromisesthe ability of muscle, fat, and other tissues to absorb glucoseproperly, leading to hyperglycemia (abnormally high levels of glucose inthe blood). Most commonly, such insulin deficiency results frominadequate production in the islet cells of the pancreas. In themajority of cases this arises from autoimmune destruction of thesecells, a condition known as type 1 or juvenile-onset diabetes, but mayalso be due to physical trauma or some other cause.

Diabetes may also arise when muscle and fat cells become less responsiveto insulin and do not absorb glucose properly, resulting inhyperglycemia. This phenomenon is known as insulin resistance, and theresulting condition is known as Type 2 diabetes. Type 2 diabetes, themost common type, is highly associated with obesity and hypertension.Obesity is associated with an inflammatory state of adipose tissue thatis thought to play a major role in the development of insulin resistance(e.g., Hotamisligil, 2006; Guilherme et al., 2008).

Diabetes is associated with damage to many tissues, largely becausehyperglycemia (and hypoglycemia, which can result from excessive orpoorly timed doses of insulin) is a significant source of oxidativestress. Chronic kidney failure, retinopathy, peripheral neuropathy,peripheral vasculitis, and the development of dermal ulcers that healslowly or not at all are among the common complications of diabetes.Because of their ability to protect against oxidative stress,particularly by the induction of HO-1 expression, compounds of theinvention may be used in treatments for many complications of diabetes.As noted above (Cai et al., 2005), chronic inflammation and oxidativestress in the liver are suspected to be primary contributing factors inthe development of Type 2 diabetes. Furthermore, PPARγ agonists such asthiazolidinediones are capable of reducing insulin resistance and areknown to be effective treatments for Type 2 diabetes.

The effect of treatment of diabetes may be evaluated as follows. Boththe biological efficacy of the treatment modality as well as theclinical efficacy are evaluated, if possible. For example, because thedisease manifests itself by increased blood sugar, the biologicalefficacy of the treatment therefore can be evaluated, for example, byobservation of return of the evaluated blood glucose towards normal.Measurement of glycosylated hemoglobin, also called A1c or HbA1c, isanother commonly used parameter of blood glucose control. Measuring aclinical endpoint which can give an indication of b-cell regenerationafter, for example, a six-month period of time, can give an indicationof the clinical efficacy of the treatment regimen.

G. Rheumatoid Arthritis

The compound and methods of this invention may be used for treatingpatients with RA. Typically the first signs of rheumatoid arthritis (RA)appear in the synovial lining layer, with proliferation of synovialfibroblasts and their attachment to the articular surface at the jointmargin (Lipsky, 1998). Subsequently, macrophages, T cells and otherinflammatory cells are recruited into the joint, where they produce anumber of mediators, including the cytokines interleukin-1 (IL-1), whichcontributes to the chronic sequelae leading to bone and cartilagedestruction, and tumour necrosis factor (TNF-α), which plays a role ininflammation (Dinarello, 1998; Arend and Dayer, 1995; van den Berg,2001). The concentration of IL-1 in plasma is significantly higher inpatients with RA than in healthy individuals and, notably, plasma IL-1levels correlate with RA disease activity (Eastgate et al., 1988).Moreover, synovial fluid levels of IL-1 are correlated with variousradiographic and histologic features of RA (Kahle et al., 1992; Rooneyet al., 1990).

In normal joints, the effects of these and other proinflammatorycytokines are balanced by a variety of anti-inflammatory cytokines andregulatory factors (Burger and Dayer, 1995). The significance of thiscytokine balance is illustrated in juvenile RA patients, who havecyclical increases in fever throughout the day (Prieur et al., 1987).After each peak in fever, a factor that blocks the effects of IL-1 isfound in serum and urine. This factor has been isolated, cloned andidentified as IL-1 receptor antagonist (IL-1ra), a member of the IL-1gene family (Hannum et al., 1990). IL-1ra, as its name indicates, is anatural receptor antagonist that competes with IL-1 for binding to typeI IL-1 receptors and, as a result, blocks the effects of IL-1 (Arend etal., 1998). A 10- to 100-fold excess of IL-1ra may be needed to blockIL-1 effectively; however, synovial cells isolated from patients with RAdo not appear to produce enough IL-1ra to counteract the effects of IL-1(Firestein et al., 1994; Fujikawa et al., 1995).

H. Psoriatic Arthritis

The compound and methods of this invention may be used for treatingpatients with psoriatic arthritis. Psoriasis is an inflammatory andproliferative skin disorder with a prevalence of 1.5-3%. Approximately20% of patients with psoriasis develop a characteristic form ofarthritis that has several patterns (Gladman, 1992; Jones et al., 1994;Gladman et al., 1995). Some individuals present with joint symptomsfirst but in the majority, skin psoriasis presents first. Aboutone-third of patients have simultaneous exacerbations of their skin andjoint disease (Gladman et al., 1987) and there is a topographicrelationship between nail and distal interphalangeal joint disease(Jones et al., 1994; Wright, 1956). Although the inflammatory processeswhich link skin, nail and joint disease remain elusive, animmune-mediated pathology is implicated.

Psoriatic arthritis (PsA) is a chronic inflammatory arthropathycharacterized by the association of arthritis and psoriasis and wasrecognized as a clinical entity distinct from rheumatoid arthritis (RA)in 1964 (Blumberg et al., 1964). Subsequent studies have revealed thatPsA shares a number of genetic, pathogenic and clinical features withother spondyloarthropathies (SpAs), a group of diseases that compriseankylosing spondylitis, reactive arthritis and enteropathic arthritis(Wright, 1979). The notion that PsA belongs to the SpA group hasrecently gained further support from imaging studies demonstratingwidespread enthesitis in the, including PsA but not RA (McGonagle etal., 1999; McGonagle et al., 1998). More specifically, enthesitis hasbeen postulated to be one of the earliest events occurring in the SpAs,leading to bone remodeling and ankylosis in the spine, as well as toarticular synovitis when the inflamed entheses are close to peripheraljoints. However, the link between enthesitis and the clinicalmanifestations in PsA remains largely unclear, as PsA can present withfairly heterogeneous patterns of joint involvement with variable degreesof severity (Marsal et al., 1999; Salvarani et al., 1998). Thus, otherfactors must be posited to account for the multifarious features of PsA,only a few of which (such as the expression of the HLA-B27 molecule,which is strongly associated with axial disease) have been identified.As a consequence, it remains difficult to map the disease manifestationsto specific pathogenic mechanisms, which means that the treatment ofthis condition remains largely empirical.

Family studies have suggested a genetic contribution to the developmentof PsA (Moll and Wright, 1973). Other chronic inflammatory forms ofarthritis, such as ankylosing spondylitis and rheumatoid arthritis, arethought to have a complex genetic basis. However, the genetic componentof PsA has been difficult to assess for several reasons. There is strongevidence for a genetic predisposition to psoriasis alone that may maskthe genetic factors that are important for the development of PsA.Although most would accept PsA as a distinct disease entity, at timesthere is a phenotypic overlap with rheumatoid arthritis and ankylosingspondylitis. Also, PsA itself is not a homogeneous condition and varioussubgroups have been proposed.

Increased amounts of TNF-α have been reported in both psoriatic skin(Ettehadi et al., 1994) and synovial fluid (Partsch et al., 1997).Recent trials have shown a positive benefit of anti-TNF treatment inboth PsA (Mease et al., 2000) and ankylosing spondylitis (Brandt et al.,2000).

I. Reactive Arthritis

The compound and methods of this invention may be used for treatingpatients with reactive arthritis. In reactive arthritis (ReA) themechanism of joint damage is unclear, but it is likely that cytokinesplay critical roles. A more prevalent Th1 profile high levels ofinterferon gamma (IFN-γ) and low levels of interleukin 4 (IL-4) has beenreported (Lahesmaa et al., 1992; Schlaak et al., 1992; Simon et al.,1993; Schlaak et al., 1996; Kotake et al., 1999; Ribbens et al., 2000),but several studies have shown relative predominance of IL-4 and IL-10and relative lack of IFN-γ and tumor necrosis factor alpha (TNF-α) inthe synovial membrane (Simon et al., 1994; Yin et al., 1999) and fluid(SF) (Yin et al., 1999; Yin et al., 1997) of reactive arthritis patientscompared with rheumatoid arthritis (RA) patients. A lower level of TNF-αsecretion in reactive arthritis than in RA patients has also beenreported after ex vivo stimulation of peripheral blood mononuclear cells(PBMC) (Braun et al., 1999).

It has been argued that clearance of reactive arthritis-associatedbacteria requires the production of appropriate levels of IFN-γ andTNF-α, while IL-10 acts by suppressing these responses (Autenrieth etal., 1994; Sieper and Braun, 1995). IL-10 is a regulatory cytokine thatinhibits the synthesis of IL-12 and TNF-γ by activated macrophages (deWaal et al., 1991; Hart et al., 1995; Chomarat et al., 1995) and ofIFN-γ by T cells (Macatonia et al., 1993).

J. Enteropathic Arthritis

The compound and methods of this invention may be used for treatingpatients with enteropathic arthritis. Typically enteropathic arthritis(EA) occurs in combination with inflammatory bowel diseases (IBD) suchas Crohn's disease or ulcerative colitis. It also can affect the spineand sacroiliac joints. Enteropathic arthritis involves the peripheraljoints, usually in the lower extremities such as the knees or ankles. Itcommonly involves only a few or a limited number of joints and mayclosely follow the bowel condition. This occurs in approximately 11% ofpatients with ulcerative colitis and 21% of those with Crohn's disease.The synovitis is generally self-limited and non-deforming.

Enteropathic arthropathies comprise a collection of rheumatologicconditions that share a link to GI pathology. These conditions includereactive (i.e., infection-related) arthritis due to bacteria (e.g.,Shigella, Salmonella, Campylobacter, Yersinia species, Clostridiumdifficile), parasites (e.g., Strongyloides stercoralis, Taenia saginata,Giardia lamblia, Ascaris lumbricoides, Cryptosporidium species), andspondyloarthropathies associated with inflammatory bowel disease (IBD).Other conditions and disorders include intestinal bypass (jejunoileal),arthritis, celiac disease, Whipple disease, and collagenous colitis.

K. Juvenile Rheumatoid Arthritis

The compound and methods of this invention may be used for treatingpatients with JRA. Juvenile rheumatoid arthritis (JRA), a term for themost prevalent form of arthritis in children, is applied to a family ofillnesses characterized by chronic inflammation and hypertrophy of thesynovial membranes. The term overlaps, but is not completely synonymous,with the family of illnesses referred to as juvenile chronic arthritisand/or juvenile idiopathic arthritis in Europe.

Both innate and adaptive immune systems use multiple cell types, a vastarray of cell surface and secreted proteins, and interconnected networksof positive and negative feedback (Lo et al., 1999). Furthermore, whileseparable in thought, the innate and adaptive wings of the immune systemare functionally intersected (Fearon and Locksley, 1996), and pathologicevents occurring at these intersecting points are likely to be highlyrelevant to our understanding of pathogenesis of adult and childhoodforms of chronic arthritis (Warrington, et al., 2001).

Polyarticular JRA is a distinct clinical subtype characterized byinflammation and synovial proliferation in multiple joints (four ormore), including the small joints of the hands (Jarvis, 2002). Thissubtype of JRA may be severe, because of both its multiple jointinvolvement and its capacity to progress rapidly over time. Althoughclinically distinct, polyarticular JRA is not homogeneous, and patientsvary in disease manifestations, age of onset, prognosis, and therapeuticresponse. These differences very likely reflect a spectrum of variationin the nature of the immune and inflammatory attack that can occur inthis disease (Jarvis, 1998).

L. Early Inflammatory Arthritis

The compound and methods of this invention may be used for treatingpatients with early inflammatory arthritis. The clinical presentation ofdifferent inflammatory arthropathies is similar early in the course ofdisease. As a result, it is often difficult to distinguish patients whoare at risk of developing the severe and persistent synovitis that leadsto erosive joint damage from those whose arthritis is more self-limited.Such distinction is critical in order to target therapy appropriately,treating aggressively those with erosive disease and avoidingunnecessary toxicity in patients with more self-limited disease. Currentclinical criteria for diagnosing erosive arthropathies such asrheumatoid arthritis (RA) are less effective in early disease andtraditional markers of disease activity such as joint counts and acutephase response do not adequately identify patients likely to have pooroutcomes (Harrison et al., 1998). Parameters reflective of thepathologic events occurring in the synovium are most likely to be ofsignificant prognostic value.

Recent efforts to identify predictors of poor outcome in earlyinflammatory arthritis have identified the presence of RA specificautoantibodies, in particular antibodies towards citrullinated peptides,to be associated with erosive and persistent disease in earlyinflammatory arthritis cohorts. On the basis of this, a cyclicalcitrullinated peptide (CCP) has been developed to assist in theidentification of anti-CCP antibodies in patient sera. Using thisapproach, the presence of anti-CCP antibodies has been shown to bespecific and sensitive for RA, can distinguish RA from otherarthropathies, and can potentially predict persistent, erosive synovitisbefore these outcomes become clinically manifest. Importantly, anti-CCPantibodies are often detectable in sera many years prior to clinicalsymptoms suggesting that they may be reflective of subclinical immuneevents (Nielen et al., 2004; Rantapaa-Dahlqvist et al., 2003).

M. Ankylosing Spondylitis

The compound and methods of this invention may be used for treatingpatients with ankylosing spondylitis. AS is a disease subset within abroader disease classification of spondyloarthropathy. Patients affectedwith the various subsets of spondyloarthropathy have disease etiologiesthat are often very different, ranging from bacterial infections toinheritance. Yet, in all subgroups, the end result of the diseaseprocess is axial arthritis. Despite the early clinically differencesseen in the various patient populations, many of them end up nearlyidentical after a disease course of ten-to-twenty years. Recent studiessuggest the mean time to clinical diagnosis of ankylosing spondylitisfrom disease onset of disease is 7.5 years (Khan, 1998). These samestudies suggest that the spondyloarthropathies may have prevalence closeto that of rheumatoid arthritis (Feldtkeller et al., 2003; Doran et al.,2003).

AS is a chronic systemic inflammatory rheumatic disorder of the axialskeleton with or without extraskeletal manifestations. Sacroiliac jointsand the spine are primarily affected, but hip and shoulder joints, andless commonly peripheral joints or certain extra-articular structuressuch as the eye, vasculature, nervous system, and gastrointestinalsystem may also be involved. Its etiology is not yet fully understood(Wordsworth, 1995; Calin and Taurog, 1998). It is strongly associatedwith the major histocompatibility class I (MHC I) HLA-B27 allele (Calinand Taurog, 1998). AS affects individuals in the prime of their life andis feared because of its potential to cause chronic pain andirreversible damage of tendons, ligaments, joints, and bones (Brewertonet al., 1973a; Brewerton et al., 1973b; Schlosstein et al., 1973). ASmay occur alone or in association with another form ofspondyloarthropathy such as reactive arthritis, psoriasis, psoriaticarthritis, enthesitis, ulcerative colitis, irritable bowel disease, orCrohn's disease, in which case it is classified as secondary AS.

Typically, the affected sites include the discovertebral, apophyseal,costovertebral, and costotransverse joints of the spine, and theparavertebral ligamentous structures. Inflammation of the entheses,which are sites of musculotendinous and ligamentous attachment to bones,is also prominent in this disease (Calin and Taurog, 1998). The site ofenthesitis is known to be infiltrated by plasma cells, lymphocytes, andpolymorphonuclear cells. The inflammatory process frequently results ingradual fibrous and bony ankylosis, (Ball, 1971; Khan, 1990).

Delayed diagnosis is common because symptoms are often attributed tomore common back problems. A dramatic loss of flexibility in the lumbarspine is an early sign of AS. Other common symptoms include chronic painand stiffness in the lower back which usually starts where the lowerspine is joined to the pelvis, or hip. Although most symptoms begin inthe lumbar and sacroiliac areas, they may involve the neck and upperback as well. Arthritis may also occur in the shoulder, hips and feet.Some patients have eye inflammation, and more severe cases must beobserved for heart valve involvement.

The most frequent presentation is back pain, but disease can beginatypically in peripheral joints, especially in children and women, andrarely with acute iritis (anterior uveitis). Additional early symptomsand signs are diminished chest expansion from diffuse costovertebralinvolvement, low-grade fever, fatigue, anorexia, weight loss, andanemia. Recurrent back pain—often nocturnal and of varying intensity—isan eventual complaint, as is morning stiffness typically relieved byactivity. A flexed or bent-over posture eases back pain and paraspinalmuscle spasm; thus, some degree of kyphosis is common in untreatedpatients.

Systemic manifestations occur in ⅓ of patients. Recurrent, usuallyself-limited, acute iritis (anterior uveitis) rarely is protracted andsevere enough to impair vision. Neurologic signs can occasionally resultfrom compression radiculitis or sciatica, vertebral fracture orsubluxation, and cauda equina syndrome (which consists of impotence,nocturnal urinary incontinence, diminished bladder and rectal sensation,and absence of ankle jerks). Cardiovascular manifestations can includeaortic insufficiency, angina, pericarditis, and ECG conductionabnormalities. A rare pulmonary finding is upper lobe fibrosis,occasionally with cavitation that may be mistaken for TB and can becomplicated by infection with Aspergillus.

AS is characterized by mild or moderate flares of active spondylitisalternating with periods of almost or totally inactive inflammation.Proper treatment in most patients results in minimal or no disabilityand in full, productive lives despite back stiffness. Occasionally, thecourse is severe and progressive, resulting in pronounced incapacitatingdeformities. The prognosis is bleak for patients with refractory iritisand for the rare patient with secondary amyloidosis.

N. Ulcerative Colitis

The compound and methods of this invention may be used for treatingpatients with ulcerative colitis. Ulcerative colitis is a disease thatcauses inflammation and sores, called ulcers, in the lining of the largeintestine. The inflammation usually occurs in the rectum and lower partof the colon, but it may affect the entire colon. Ulcerative colitisrarely affects the small intestine except for the end section, calledthe terminal ileum. Ulcerative colitis may also be called colitis orproctitis. The inflammation makes the colon empty frequently, causingdiarrhea. Ulcers form in places where the inflammation has killed thecells lining the colon; the ulcers bleed and produce pus.

Ulcerative colitis is an inflammatory bowel disease (IBD), the generalname for diseases that cause inflammation in the small intestine andcolon. Ulcerative colitis can be difficult to diagnose because itssymptoms are similar to other intestinal disorders and to another typeof IBD, Crohn's disease. Crohn's disease differs from ulcerative colitisbecause it causes inflammation deeper within the intestinal wall. Also,Crohn's disease usually occurs in the small intestine, although it canalso occur in the mouth, esophagus, stomach, duodenum, large intestine,appendix, and anus.

Ulcerative colitis may occur in people of any age, but most often itstarts between ages 15 and 30, or less frequently between ages 50 and70. Children and adolescents sometimes develop the disease. Ulcerativecolitis affects men and women equally and appears to run in somefamilies. Theories about what causes ulcerative colitis abound, but nonehave been proven. The most popular theory is that the body's immunesystem reacts to a virus or a bacterium by causing ongoing inflammationin the intestinal wall. People with ulcerative colitis haveabnormalities of the immune system, but doctors do not know whetherthese abnormalities are a cause or a result of the disease. Ulcerativecolitis is not caused by emotional distress or sensitivity to certainfoods or food products, but these factors may trigger symptoms in somepeople.

The most common symptoms of ulcerative colitis are abdominal pain andbloody diarrhea. Patients also may experience fatigue, weight loss, lossof appetite, rectal bleeding, and loss of body fluids and nutrients.About half of patients have mild symptoms. Others suffer frequent fever,bloody diarrhea, nausea, and severe abdominal cramps. Ulcerative colitismay also cause problems such as arthritis, inflammation of the eye,liver disease (hepatitis, cirrhosis, and primary sclerosingcholangitis), osteoporosis, skin rashes, and anemia. No one knows forsure why problems occur outside the colon. Scientists think thesecomplications may occur when the immune system triggers inflammation inother parts of the body. Some of these problems go away when the colitisis treated.

A thorough physical exam and a series of tests may be required todiagnose ulcerative colitis. Blood tests may be done to check foranemia, which could indicate bleeding in the colon or rectum. Bloodtests may also uncover a high white blood cell count, which is a sign ofinflammation somewhere in the body. By testing a stool sample, thedoctor can detect bleeding or infection in the colon or rectum. Thedoctor may do a colonoscopy or sigmoidoscopy. For either test, thedoctor inserts an endoscope—a long, flexible, lighted tube connected toa computer and TV monitor—into the anus to see the inside of the colonand rectum. The doctor will be able to see any inflammation, bleeding,or ulcers on the colon wall. During the exam, the doctor may do abiopsy, which involves taking a sample of tissue from the lining of thecolon to view with a microscope. A barium enema x-ray of the colon mayalso be required. This procedure involves filling the colon with barium,a chalky white solution. The barium shows up white on x-ray film,allowing the doctor a clear view of the colon, including any ulcers orother abnormalities that might be there.

Treatment for ulcerative colitis depends on the seriousness of thedisease. Most people are treated with medication. In severe cases, apatient may need surgery to remove the diseased colon. Surgery is theonly cure for ulcerative colitis. Some people whose symptoms aretriggered by certain foods are able to control the symptoms by avoidingfoods that upset their intestines, like highly seasoned foods, rawfruits and vegetables, or milk sugar (lactose). Each person mayexperience ulcerative colitis differently, so treatment is adjusted foreach individual. Emotional and psychological support is important. Somepeople have remissions—periods when the symptoms go away—that last formonths or even years. However, most patients' symptoms eventuallyreturn. This changing pattern of the disease means one cannot alwaystell when a treatment has helped. Some people with ulcerative colitismay need medical care for some time, with regular doctor visits tomonitor the condition.

O. Crohn's Disease

The compound and methods of this invention may be used for treatingpatients with Crohn's disease. Another disorder for whichimmunosuppression has been tried is Crohn's disease. Crohn's diseasesymptoms include intestinal inflammation and the development ofintestinal stenosis and fistulas; neuropathy often accompanies thesesymptoms. Anti-inflammatory drugs, such as 5-aminosalicylates (e.g.,mesalamine) or corticosteroids, are typically prescribed, but are notalways effective (reviewed in Botoman et al., 1998). Immunosuppressionwith cyclosporine is sometimes beneficial for patients resistant to orintolerant of corticosteroids (Brynskov et al., 1989).

Efforts to develop diagnostic and treatment tools against Crohn'sdisease have focused on the central role of cytokines (Schreiber, 1998;van Hogezand and Verspaget, 1998). Cytokines are small secreted proteinsor factors (5 to 20 kD) that have specific effects on cell-to-cellinteractions, intercellular communication, or the behavior of othercells. Cytokines are produced by lymphocytes, especially TH1 and TH2lymphocytes, monocytes, intestinal macrophages, granulocytes, epithelialcells, and fibroblasts (reviewed in Rogler and Andus, 1998; Galley andWebster, 1996). Some cytokines are pro-inflammatory (e.g., TNF-α, IL-1(αand β), IL-6, IL-8, IL-12, or leukemia inhibitory factor [LIF]); othersare anti-inflammatory (e.g., IL-1 receptor antagonist, IL-4, IL-10,IL-11, and TGF-β). However, there may be overlap and functionalredundancy in their effects under certain inflammatory conditions.

In active cases of Crohn's disease, elevated concentrations of TNF-α andIL-6 are secreted into the blood circulation, and TNF-α, IL-1, IL-6, andIL-8 are produced in excess locally by mucosal cells (id.; Funakoshi etal., 1998). These cytokines can have far-ranging effects onphysiological systems including bone development, hematopoiesis, andliver, thyroid, and neuropsychiatric function. Also, an imbalance of theIL-1β/IL-1ra ratio, in favor of pro-inflammatory IL-1β, has beenobserved in patients with Crohn's disease (Rogler and Andus, 1998; Saikiet al., 1998; Dionne et al., 1998; but see Kuboyama, 1998). One studysuggested that cytokine profiles in stool samples could be a usefuldiagnostic tool for Crohn's disease (Saiki et al., 1998).

Treatments that have been proposed for Crohn's disease include the useof various cytokine antagonists (e.g., IL-1ra), inhibitors (e.g., ofIL-1(3 converting enzyme and antioxidants) and anti-cytokine antibodies(Rogler and Andus, 1998; van Hogezand and Verspaget, 1998; Reimund etal., 1998; Lugering et al., 1998; McAlindon et al., 1998). Inparticular, monoclonal antibodies against TNF-α have been tried withsome success in the treatment of Crohn's disease (Targan et al., 1997;Stack et al., 1997; van Dullemen et al., 1995). These compounds may beused in combination therapy with compounds of the present disclosure.

Another approach to the treatment of Crohn's disease has focused on atleast partially eradicating the bacterial community that may betriggering the inflammatory response and replacing it with anon-pathogenic community. For example, U.S. Pat. No. 5,599,795 disclosesa method for the prevention and treatment of Crohn's disease in humanpatients. Their method was directed to sterilizing the intestinal tractwith at least one antibiotic and at least one anti-fungal agent to killoff the existing flora and replacing them with different, select,well-characterized bacteria taken from normal humans. Borody taught amethod of treating Crohn's disease by at least partial removal of theexisting intestinal microflora by lavage and replacement with a newbacterial community introduced by fecal inoculum from a disease-screenedhuman donor or by a composition comprising Bacteroides and Escherichiacoli species. (U.S. Pat. No. 5,443,826).

P. Systemic Lupus Erythematosus

The compound and methods of this invention may be used for treatingpatients with SLE. There has also been no known cause for autoimmunediseases such as systemic lupus erythematosus. Systemic lupuserythematosus (SLE) is an autoimmune rheumatic disease characterized bydeposition in tissues of autoantibodies and immune complexes leading totissue injury (Kotzin, 1996). In contrast to autoimmune diseases such asMS and type 1 diabetes mellitus, SLE potentially involves multiple organsystems directly, and its clinical manifestations are diverse andvariable (reviewed by Kotzin and O'Dell, 1995). For example, somepatients may demonstrate primarily skin rash and joint pain, showspontaneous remissions, and require little medication. At the other endof the spectrum are patients who demonstrate severe and progressivekidney involvement that requires therapy with high doses of steroids andcytotoxic drugs such as cyclophosphamide (Kotzin, 1996).

The serological hallmark of SLE, and the primary diagnostic testavailable, is elevated serum levels of IgG antibodies to constituents ofthe cell nucleus, such as double-stranded DNA (dsDNA), single-strandedDNA (ss-DNA), and chromatin. Among these autoantibodies, IgG anti-dsDNAantibodies play a major role in the development of lupusglomerulonephritis (G N) (Hahn and Tsao, 1993; Ohnishi et al., 1994).Glomerulonephritis is a serious condition in which the capillary wallsof the kidney's blood purifying glomeruli become thickened by accretionson the epithelial side of glomerular basement membranes. The disease isoften chronic and progressive and may lead to eventual renal failure.

Q. Irritable Bowel Syndrome

The compound and methods of this invention may be used for treatingpatients with Irritable bowel syndrome (IBS). IBS is a functionaldisorder characterized by abdominal pain and altered bowel habits. Thissyndrome may begin in young adulthood and can be associated withsignificant disability. This syndrome is not a homogeneous disorder.Rather, subtypes of IBS have been described on the basis of thepredominant symptom—diarrhea, constipation, or pain. In the absence of“alarm” symptoms, such as fever, weight loss, and gastrointestinalbleeding, a limited workup is needed. Once a diagnosis of IBS is made,an integrated treatment approach can effectively reduce the severity ofsymptoms. IBS is a common disorder, although its prevalence rates havevaried. In general, IBS affects about 15% of US adults and occurs aboutthree times more often in women than in men (Jailwala et al., 2000).

IBS accounts for between 2.4 million and 3.5 million visits tophysicians each year. It not only is the most common condition seen bygastroenterologists but also is one of the most common gastrointestinalconditions seen by primary care physicians (Everhart et al., 1991;Sandler, 1990).

IBS is also a costly disorder. Compared with persons who do not havebowel symptoms, persons with IBS miss three times as many workdays andare more likely to report being too sick to work (Drossman et al., 1993;Drossman et al., 1997). Moreover, those with IBS incur hundreds ofdollars more in medical charges than persons without bowel disorders(Talley et al., 1995).

No specific abnormality accounts for the exacerbations and remissions ofabdominal pain and altered bowel habits experienced by patients withIBS. The evolving theory of IBS suggests dysregulation at multiplelevels of the brain-gut axis. Dysmotility, visceral hypersensitivity,abnormal modulation of the central nervous system (CNS), and infectionhave all been implicated. In addition, psychosocial factors play animportant modifying role. Abnormal intestinal motility has long beenconsidered a factor in the pathogenesis of IBS. Transit time through thesmall intestine after a meal has been shown to be shorter in patientswith diarrhea-predominant IBS than in patients who have theconstipation-predominant or pain-predominant subtype (Cann et al.,1983).

In studies of the small intestine during fasting, the presence of bothdiscrete, clustered contractions and prolonged, propagated contractionshas been reported in patients with IBS (Kellow and Phillips, 1987). Theyalso experience pain with irregular contractions more often than healthypersons (Kellow and Phillips, 1987; Horwitz and Fisher, 2001) Thesemotility findings do not account for the entire symptom complex inpatients with IBS; in fact, most of these patients do not havedemonstrable abnormalities (Rothstein, 2000). Patients with IBS haveincreased sensitivity to visceral pain. Studies involving balloondistention of the rectosigmoid colon have shown that patients with IBSexperience pain and bloating at pressures and volumes much lower thancontrol subjects (Whitehead et al., 1990). These patients maintainnormal perception of somatic stimuli.

Multiple theories have been proposed to explain this phenomenon. Forexample, receptors in the viscera may have increased sensitivity inresponse to distention or intraluminal contents. Neurons in the dorsalhorn of the spinal cord may have increased excitability. In addition,alteration in CNS processing of sensations may be involved (Drossman etal., 1997). Functional magnetic resonance imaging studies have recentlyshown that compared with control subjects, patients with IBS haveincreased activation of the anterior cingulate cortex, an important paincenter, in response to a painful rectal stimulus (Mertz et al., 2000).

Increasingly, evidence suggests a relationship between infectiousenteritis and subsequent development of IBS. Inflammatory cytokines mayplay a role. In a survey of patients with a history of confirmedbacterial gastroenteritis (Neal et al., 1997), 25% reported persistentalteration of bowel habits. Persistence of symptoms may be due topsychological stress at the time of acute infection (Gwee et al., 1999).

Recent data suggest that bacterial overgrowth in the small intestine mayhave a role in IBS symptoms. In one study (Pimentel et al., 2000), 157(78%) of 202 IBS patients referred for hydrogen breath testing had testfindings that were positive for bacterial overgrowth. Of the 47 subjectswho had follow-up testing, 25 (53%) reported improvement in symptoms(i.e., abdominal pain and diarrhea) with antibiotic treatment.

IBS may present with a range of symptoms. However, abdominal pain andaltered bowel habits remain the primary features. Abdominal discomfortis often described as crampy in nature and located in the left lowerquadrant, although the severity and location can differ greatly.Patients may report diarrhea, constipation, or alternating episodes ofdiarrhea and constipation. Diarrheal symptoms are typically described assmall-volume, loose stools, and stool is sometimes accompanied by mucusdischarge. Patients also may report bloating, fecal urgency, incompleteevacuation, and abdominal distention. Upper gastrointestinal symptoms,such as gastroesophageal reflux, dyspepsia, or nausea, may also bepresent (Lynn and Friedman, 1993).

Persistence of symptoms is not an indication for further testing; it isa characteristic of IBS and is itself an expected symptom of thesyndrome. More extensive diagnostic evaluation is indicated in patientswhose symptoms are worsening or changing. Indications for furthertesting also include presence of alarm symptoms, onset of symptoms afterage 50, and a family history of colon cancer. Tests may includecolonoscopy, computed tomography of the abdomen and pelvis, and bariumstudies of the small or large intestine.

R. Sjögren's Syndrome

The compound and methods of this invention may be used for treatingpatients with Sjögren's syndrome. Primary Sjögren's syndrome (SS) is achronic, slowly progressive, systemic autoimmune disease, which affectspredominantly middle-aged women (female-to-male ratio 9:1), although itcan be seen in all ages including childhood (Jonsson et al., 2002). Itis characterized by lymphocytic infiltration and destruction of theexocrine glands, which are infiltrated by mononuclear cells includingCD4+, CD8+ lymphocytes and B-cells (Jonsson et al., 2002). In addition,extraglandular (systemic) manifestations are seen in one-third ofpatients (Jonsson et al., 2001).

The glandular lymphocytic infiltration is a progressive feature (Jonssonet al., 1993), which, when extensive, may replace large portions of theorgans. Interestingly, the glandular infiltrates in some patientsclosely resemble ectopic lymphoid microstructures in the salivary glands(denoted as ectopic germinal centers) (Salomonsson et al., 2002; Xanthouet al., 2001). In SS, ectopic GCs are defined as T and B cell aggregatesof proliferating cells with a network of follicular dendritic cells andactivated endothelial cells. These GC-like structures formed within thetarget tissue also portray functional properties with production ofautoantibodies (anti-Ro/SSA and anti-La/SSB) (Salomonsson and Jonsson,2003).

In other systemic autoimmune diseases, such as RA, factors critical forectopic GCs have been identified. Rheumatoid synovial tissues with GCswere shown to produce chemokines CXCL13, CCL21 and lymphotoxin (LT)-β(detected on follicular center and mantle zone B cells). Multivariateregression analysis of these analytes identified CXCL13 and LT-β as thesolitary cytokines predicting GCs in rheumatoid synovitis (Weyand andGoronzy, 2003). Recently CXCL13 and CXCR5 in salivary glands has beenshown to play an essential role in the inflammatory process byrecruiting B and T cells, therefore contributing to lymphoid neogenesisand ectopic GC formation in SS (Salomonsson et al., 2002).

S. Psoriasis

The compound and methods of this invention may be used for treatingpatients with psoriasis. Psoriasis is a chronic skin disease of scalingand inflammation that affects 2 to 2.6 percent of the United Statespopulation, or between 5.8 and 7.5 million people. Although the diseaseoccurs in all age groups, it primarily affects adults. It appears aboutequally in males and females. Psoriasis occurs when skin cells quicklyrise from their origin below the surface of the skin and pile up on thesurface before they have a chance to mature. Usually this movement (alsocalled turnover) takes about a month, but in psoriasis it may occur inonly a few days. In its typical form, psoriasis results in patches ofthick, red (inflamed) skin covered with silvery scales. These patches,which are sometimes referred to as plaques, usually itch or feel sore.They most often occur on the elbows, knees, other parts of the legs,scalp, lower back, face, palms, and soles of the feet, but they canoccur on skin anywhere on the body. The disease may also affect thefingernails, the toenails, and the soft tissues of the genitals andinside the mouth. While it is not unusual for the skin around affectedjoints to crack, approximately 1 million people with psoriasisexperience joint inflammation that produces symptoms of arthritis. Thiscondition is called psoriatic arthritis.

Psoriasis is a skin disorder driven by the immune system, especiallyinvolving a type of white blood cell called a T cell. Normally, T cellshelp protect the body against infection and disease. In the case ofpsoriasis, T cells are put into action by mistake and become so activethat they trigger other immune responses, which lead to inflammation andto rapid turnover of skin cells. In about one-third of the cases, thereis a family history of psoriasis. Researchers have studied a largenumber of families affected by psoriasis and identified genes linked tothe disease. People with psoriasis may notice that there are times whentheir skin worsens, then improves. Conditions that may cause flareupsinclude infections, stress, and changes in climate that dry the skin.Also, certain medicines, including lithium and beta blockers, which areprescribed for high blood pressure, may trigger an outbreak or worsenthe disease.

T. Infectious Diseases

Compound of the present disclosure may be useful in the treatment ofinfectious diseases, including viral and bacterial infections. As notedabove, such infections may be associated with severe localized orsystemic inflammatory responses. For example, influenza may cause severeinflammation of the lung and bacterial infection can cause the systemichyperinflammatory response, including the excessive production ofmultiple inflammatory cytokines, that is the hallmark of sepsis. Inaddition, compounds of the invention may be useful in directlyinhibiting the replication of viral pathogens. Previous studies havedemonstrated that related compounds such as CDDO can inhibit thereplication of HIV in macrophages (Vazquez et al., 2005). Other studieshave indicated that inhibition of NF-kappa B signaling may inhibitinfluenza virus replication, and that cyclopentenone prostaglandins mayinhibit viral replication (e.g., Mazur et al., 2007; Pica et al., 2000).

The present invention relates to the treatment or prevention of each ofthe diseases/disorders/conditions referred to above in section IV usingthe compound RTA 408 or a pharmaceutically acceptable salt thereof, or apolymorphic form of that compound (such as, e.g., any one of thepolymorphic forms described herein above or below), or a pharmaceuticalcomposition comprising any of the aforementioned entities and apharmaceutically acceptable carrier (including, e.g., the pharmaceuticalcompositions described herein above or below).

V. PHARMACEUTICAL FORMULATIONS AND ROUTES OF ADMINISTRATION

RTA 408 may be administered by a variety of methods, e.g., orally or byinjection (e.g., subcutaneous, intravenous, intraperitoneal, etc.).Depending on the route of administration, the active compounds may becoated in a material to protect the compound from the action of acidsand other natural conditions which may inactivate the compound. They mayalso be administered by continuous perfusion/infusion of a disease orwound site.

To administer RTA 408 by other than parenteral administration, it may benecessary to coat the compound with, or co-administer the compound with,a material to prevent its inactivation. For example, the therapeuticcompound may be administered to a patient in an appropriate carrier, forexample, liposomes, or a diluent. Pharmaceutically acceptable diluentsinclude saline and aqueous buffer solutions. Liposomes includewater-in-oil-in-water CGF emulsions as well as conventional liposomes(Strejan et al., 1984).

RTA 408 may also be administered parenterally, intraperitoneally,intraspinally, or intracerebrally. Dispersions can be prepared inglycerol, liquid polyethylene glycols, and mixtures thereof and in oils.Under ordinary conditions of storage and use, these preparations maycontain a preservative to prevent the growth of microorganisms.

Sterile injectable solutions can be prepared by incorporating RTA 408 inthe required amount in an appropriate solvent with one or a combinationof ingredients enumerated above, as required, followed by filteredsterilization. Generally, dispersions are prepared by incorporating thetherapeutic compound into a sterile carrier that contains a basicdispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying, which yields a powder of the activeingredient (i.e., the therapeutic compound) plus any additional desiredingredient from a previously sterile-filtered solution thereof.

RTA 408 may be rendered fully amorphous using a direct spray dryingprocedure. RTA 408 can be orally administered, for example, with aninert diluent or an assimilable edible carrier. The therapeutic compoundand other ingredients may also be enclosed in a hard or soft shellgelatin capsule, compressed into tablets, or incorporated directly intothe patient's diet. For oral therapeutic administration, the therapeuticcompound may be incorporated with excipients and used in the form ofingestible tablets, buccal tablets, troches, capsules, elixirs,suspensions, syrups, wafers, and the like. The percentage of thetherapeutic compound in the compositions and preparations may, ofcourse, be varied. The amount of the therapeutic compound in suchtherapeutically useful compositions is such that a suitable dosage willbe obtained.

It is especially advantageous to formulate parenteral compositions indosage unit form for ease of administration and uniformity of dosage.Dosage unit form as used herein refers to physically discrete unitssuited as unitary dosages for the patients to be treated, each unitcontaining a predetermined quantity of therapeutic compound calculatedto produce the desired therapeutic effect in association with therequired pharmaceutical carrier. The specification for the dosage unitforms of the invention are dictated by and directly dependent on (a) theunique characteristics of the therapeutic compound and the particulartherapeutic effect to be achieved, and (b) the limitations inherent inthe art of compounding such a therapeutic compound for the treatment ofa selected condition in a patient.

RTA 408 may also be administered topically to the skin, eye, or mucosa.Alternatively, if local delivery to the lungs is desired the therapeuticcompound may be administered by inhalation in a dry-powder or aerosolformulation.

RTA 408 will typically be administered at a therapeutically effectivedosage sufficient to treat a condition associated with a given patient.For example, the efficacy of a compound can be evaluated in an animalmodel system that may be predictive of efficacy in treating the diseasein humans, such as the model systems shown in the examples and drawings.

The actual dosage amount of RTA 408 or composition comprising RTA 408administered to a patient may be determined by physical andphysiological factors, such as age, sex, body weight, severity ofcondition, the type of disease being treated, previous or concurrenttherapeutic interventions, idiopathy of the patient, and the route ofadministration. These factors may be determined by a skilled artisan.The practitioner responsible for administration will typically determinethe concentration of active ingredient(s) in a composition andappropriate dose(s) for the individual patient. The dosage may beadjusted by the individual physician in the event of any complication.

An effective amount typically will vary from about 0.001 mg/kg to about1000 mg/kg, from about 0.01 mg/kg to about 750 mg/kg, from about 100mg/kg to about 500 mg/kg, from about 1.0 mg/kg to about 250 mg/kg, fromabout 10.0 mg/kg to about 150 mg/kg in one or more dose administrationsdaily, for one or several days (depending of course of the mode ofadministration and the factors discussed above). Other suitable doseranges include 1 mg to 10000 mg per day, 100 mg to 10000 mg per day, 500mg to 10000 mg per day, and 500 mg to 1000 mg per day. In someparticular embodiments, the amount is less than 10,000 mg per day with arange of 750 mg to 9000 mg per day.

The effective amount may be less than 1 mg/kg/day, less than 500mg/kg/day, less than 250 mg/kg/day, less than 100 mg/kg/day, less than50 mg/kg/day, less than 25 mg/kg/day, or less than 10 mg/kg/day. It mayalternatively be in the range of 1 mg/kg/day to 200 mg/kg/day. In someembodiments, the amount could be 10, 30, 100, or 150 mg/kg formulated asa suspension in sesame oil. In some embodiments, the amount could be 3,10, 30 or 100 mg/kg administered daily via oral gavage. In someembodiments, the amount could be 10, 30, or 100 mg/kg administeredorally. For example, regarding treatment of diabetic patients, the unitdosage may be an amount that reduces blood glucose by at least 40% ascompared to an untreated patient. In another embodiment, the unit dosageis an amount that reduces blood glucose to a level that is ±10% of theblood glucose level of a non-diabetic patient.

In other non-limiting examples, a dose may also comprise from about 1micro-gram/kg/body weight, about 5 microgram/kg/body weight, about 10microgram/kg/body weight, about 50 microgram/kg/body weight, about 100microgram/kg/body weight, about 200 microgram/kg/body weight, about 350microgram/kg/body weight, about 500 microgram/kg/body weight, about 1milligram/kg/body weight, about 5 milligram/kg/body weight, about 10milligram/kg/body weight, about 50 milligram/kg/body weight, about 100milligram/kg/body weight, about 200 milligram/kg/body weight, about 350milligram/kg/body weight, about 500 milligram/kg/body weight, to about1000 mg/kg/body weight or more per administration, and any rangederivable therein. In non-limiting examples of a derivable range fromthe numbers listed herein, a range of about 5 mg/kg/body weight to about100 mg/kg/body weight, about 5 microgram/kg/body weight to about 500milligram/kg/body weight, etc., can be administered, based on thenumbers described above.

In certain embodiments, a pharmaceutical composition of the presentdisclosure may comprise, for example, at least about 0.01% of RTA 408.In other embodiments, RTA 408 may comprise between about 0.01% to about75% of the weight of the unit, or between about 0.01% to about 5%, forexample, and any range derivable therein. In some embodiments, RTA 408may be used in a formulation such as a suspension in sesame oil of 0.01,0.1, or 1%.

Single or multiple doses of the agent comprising RTA 408 arecontemplated. Desired time intervals for delivery of multiple doses canbe determined by one of ordinary skill in the art employing no more thanroutine experimentation. As an example, patients may be administered twodoses daily at approximately 12 hour intervals. In some embodiments, theagent is administered once a day. The agent(s) may be administered on aroutine schedule. As used herein a routine schedule refers to apredetermined designated period of time. The routine schedule mayencompass periods of time that are identical or that differ in length,as long as the schedule is predetermined. For instance, the routineschedule may involve administration twice a day, every day, every twodays, every three days, every four days, every five days, every sixdays, a weekly basis, a monthly basis, or any set number of days orweeks there-between. Alternatively, the predetermined routine schedulemay involve administration on a twice daily basis for the first week,followed by a daily basis for several months, etc. In other embodiments,the invention provides that the agent(s) may be taken orally and thatthe timing of which is or is not dependent upon food intake. Thus, forexample, the agent can be taken every morning and/or every evening,regardless of when the patient has eaten or will eat.

VI. EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

A. Synthesis of RTA 408 (63415)

Compound 1: RTA 401 (20.0 g, 40.6 mmol), triethylamine (17.0 mL, 122.0mmol), and toluene (400 mL) were added into a reactor and cooled to 0°C. with stirring. Diphenyl phosphoryl azide (DPPA) (13.2 mL, 61.0 mmol)was added with stirring at 0° C. over 5 minutes, and the mixture wascontinually stirred at room temperature overnight (HPLC-MS check showsno RTA 401 left). The reaction mixture was directly loaded on a silicagel column and purified by column chromatography (silica gel, 0% to 5%ethyl acetate in CH₂Cl₂) to give compound 1 (19.7 g, ˜94%, partiallyconverted into compound 2) as a white foam.

Compound 2: Compound 1 (19.7 g, ˜38.1 mmol) and benzene (250 mL) wereadded into a reactor and heated to 80° C. with stirring for 2 hours(HPLC-MS check shows no compound 1 left). The reaction mixture wasconcentrated at reduced pressure to afford crude compound 2 as a solidresidue, which was used for the next step without purification.

Compound 3: Crude compound 2 (≤38.1 mmol) and CH₃CN (200 mL) were addedinto a reactor and cooled to 0° C. with stirring. HCl (12 N, 90 mL) wasadded at 0° C. over 1 minute, and the mixture was continually stirred atroom temperature for 1 hour (HPLC-MS check shows no compound 2 left).The reaction mixture was cooled to 0° C. and 10% NaOH (˜500 mL) wasadded with stirring. Then, saturated NaHCO₃(1 L) was added withstirring. The aqueous phase was extracted by ethyl acetate (2×500 mL).The combined organic phase was washed by H₂O (200 mL), saturated NaCl(200 mL), dried over Na₂SO₄, and concentrated to afford crude compound 3(16.62 g) as a light yellow foam, which was used for the next stepwithout purification.

RTA 408: Crude amine 3 (16.62 g, 35.9 mmol), CH₃CF₂CO₂H (4.7388 g, 43.1mmol), and CH₂Cl₂ (360 mL) were added into a reactor with stirring atroom temperature. Then, dicyclohexylcarbodiimide (DCC) (11.129 g, 53.9mmol) and 4-(dimethylamino)pyridine (DMAP) (1.65 g, 13.64 mmol) wereadded and the mixture was continually stirred at room temperatureovernight (HPLC-MS check shows no compound 3 left). The reaction mixturewas filtered to remove solid by-products, and the filtrate was directlyloaded on a silica gel column and purified by column chromatography(silica gel, 0% to 20% ethyl acetate in hexanes) twice to give compoundRTA 408 (16.347 g, 73% from RTA 401 over 4 steps) as a white foam: ¹HNMR (400 MHz, CD₃Cl) δ ppm 8.04 (s, 1H), 6.00 (s, 1H), 5.94 (s, br, 1H),3.01 (d, 1H, J=4.8 Hz), 2.75-2.82 (m, 1H), 1.92-2.18 (m, 4H), 1.69-1.85(m, 7H), 1.53-1.64 (m, 1H), 1.60 (s, 3H), 1.50 (s, 3H), 1.42 (s, 3H),1.11-1.38 (m, 3H), 1.27 (s, 3H), 1.18 (s, 3H), 1.06 (s, 3H), 1.04 (s,3H), 0.92 (s, 3H); m/z 555 (M+1).

B. Pharmacodynamics

A summary of the in vitro and in vivo studies to evaluate the primarypharmacodynamic effects of RTA 408 is provided below.

1. Effects of RTA 408 on Keap1-Nrf2 and NF-κB in Vitro

Inhibition of IFNγ-induced NO production by AIMs is Nrf2-dependent(Dinkova-Kostova, 2005). RAW264.7 mouse macrophages were pre-treatedwith dimethyl sulfoxide (vehicle) or RTA 408 for 2 hours, followed bytreatment with 20 ng/mL of mouse IFNγ for 24 hours. Nitrite (NO₂ ⁻)levels in the media were measured as a surrogate for nitric oxide usingthe Griess reagent assay. Cell viability was assessed using the WST-1assay. Treatment with RTA 408 resulted in a dose-dependent suppressionof IFNγ-induced NO production, with an average IC₅₀ value of 3.8±1.2 nM.Results from a representative experiment are shown in FIG. 1. The IC₅₀value for RTA 408 was found 45%-65% lower than the IC₅₀ values forcompounds 63170 (8±3 nM), 63171 (6.9±0.6 nM), 63179 (11±2 nm), and 63189(7±2 nM). 63170, 63171, 63179, and 63189 are compounds of the formulas:

2. Effect of RTA 408 on Nrf2 Target Genes

RTA 408 was tested in two different reporter assays to assess activationof the antioxidant response element (ARE). The first reporter tested wascontrolled by an ARE derived from the human NQO1 gene. The HuH-7 humanhepatoma cell line was transiently transfected with an NQO1-AREluciferase reporter plasmid, and cells were treated with RTA 408 for 18hours. FIG. 2a shows a dose-dependent induction of luciferase activityby RTA 408 in this cell line. Values represent the average of threeindependent experiments. Twenty percent less RTA 408 (12 nM) than 63189(14.9 nM) was required to increase transcription from the NQO1 ARE inHuH-7 cells by 2-fold. Likewise, 2.1-2.4 fold less RTA 408 than 63170(25.2 nM) and 63179 (29.1 nM), respectively, was required to increasetranscription from the NQO1 ARE in HuH-7 cells by 2-fold. The effect ofRTA 408 on luciferase reporter activation was also assessed in theAREc32 reporter cell line. This cell line is derived from human breastcarcinoma MCF-7 cells and is stably transfected with a luciferasereporter gene under the transcriptional control of eight copies of therat GSTA2 ARE sequence. Following treatment with RTA 408 for 18 hours, asimilar dose-dependent response was observed in the AREc32 reporter cellline (FIG. 2b ). An ˜2-fold induction of luciferase activity was evidentfollowing treatment with 15.6 nM RTA 408 in both reporter assays.

RTA 408 was also shown to increase transcript levels of known Nrf2target genes in the HFL1 human lung fibroblast and BEAS-2B humanbronchial epithelial cell lines. Treatment of HFL1 lung fibroblasts withRTA 408 for 18 hours resulted in increased expression of several Nrf2target genes, including NQO1, HMOX1, GCLM, and TXNRD1, as measured byquantitative PCR (FIGS. 3a-d ). For all genes tested, induction by RTA408 was dose-dependent and evident at concentrations as low as 15.6 nM.Treatment of BEAS-2B bronchial epithelial cells with RTA 408 for 18hours resulted in a similar dose-dependent increase of all Nrf2 targetgenes evaluated (FIGS. 4a-d ). RTA 408 also increased expression of Nrf2target genes in normal human mesangial cells (nHMC), the mouse BV2microglial cell line, and the human SH-SY5Y neuroblastoma cell line atsimilar concentrations.

Treatment with RTA 408 also increased NQO1 protein levels in SH-SY5Ycells in a dose-dependent manner (FIG. 5a ). HMOX1 protein was notdetected in untreated or RTA 408-treated SH-SY5Y cells. In BV2 cells,treatment with RTA 408 increased NQO1 and HMOX1 protein levels atconcentrations up to 125 nM (FIG. 5b ). The EC₅₀ value for induction ofNrf2 protein expression in SK-N-SH cells by RTA 408 (56.4 nM) was45%-65% lower than the EC₅₀ values for 63171 (122 nM), 63189 (102 nM),and 63179 (126 nM). The same amount of 63170 (54.6 nM) was required.

The EC₅₀ was measured using an in-cell western NQO1 assay where thecells were incubated with the compound under evaluation for 3 days.After incubation with the compound of interest, the cells were reactedwith mouse NQO1 antibody and then the next day the cells were reactedwith IRDye-800CW-anti-mouse IgG antibody. The target signals werevisualized and then analyzed.

Consistent with induction of Nrf2 target genes and corresponding proteinproducts, treatment of RAW264.7 mouse macrophage cells for 24 hoursincreased NQO1 enzymatic activity in a dose-dependent manner, withincreases evident at 7.8 nM (FIG. 6).

Taken together, these data from multiple cell lines demonstrate thattreatment with RTA 408 increases transcriptional activity controlled byantioxidant response elements, increases expression of Nrf2 targetgenes, and increases the activity of NQO1, an Nrf2 target gene product.

3. Effect of RTA 408 on Markers of Cellular Redox Capacity

Glutathione and NADPH are critical factors required for the maintenanceof cellular redox capacity. Several genes involved in the synthesis ofglutathione (e.g., GCLC and GLCM) and NADPH [e.g., hexose-6-phosphatedehydrogenase (H6PD) and malic enzyme 1 (ME1)] have been demonstrated tobe regulated by Nrf2 (Wu, 2011). The effect of RTA 408 treatment ontotal glutathione levels was evaluated in the mouse AML-12 hepatocytecell line. Treatment of AML-12 cells for 24 hours with RTA 408 increasedtotal cellular glutathione levels in a dose-dependent manner (FIG. 7).Data shown are representative of two independent experiments. A >2-foldincrease in total glutathione was observed at RTA 408 concentrations aslow as 15.6 nM. The EC₅₀ value using a RAW264.7 mouse model forinduction of glutathione levels by RTA 408 (9.9 nM) was 22%-57% lowerthan the EC₅₀ values for 63170 (12.1 nM), 63171 (23.2 nM), and 63189 (16nM).

The effect of RTA 408 treatment on the levels of NADPH, as measured bythe absorbance of a redox-sensitive dye, WST-1, was evaluated in HCT-116cells. RTA 408 treatment for 24 hours increased WST-1 absorbance in adose-dependent manner (FIG. 8), suggesting that NADPH levels wereincreased.

The effect of RTA 408 on the expression of genes involved in NADPHsynthesis pathways was also evaluated in this study. HCT-116 cells weretreated with RTA 408 for 24 hours, and mRNA levels of H6PD,phosphogluconate dehydrogenase (PGD), transketolase (TKT), and ME1 weremeasured using quantitative PCR. Treatment with RTA 408 resulted in adose-dependent increase in expression of genes involved in NADPHsynthesis (FIGS. 9a-d ).

In summary, treatment with RTA 408 increased total glutathione levels inAML-12 hepatocytes and increased WST-1 absorbance, a marker of NADPHproduction, in HCT-116 cells. This observation correlated with anincrease in the expression of several key genes encoding enzymesinvolved in NADPH synthesis.

4. Effect of RTA 408 on TNFα-induced NF-κB Signaling

NF-κB is a transcription factor that plays a central role in theregulation of many immune and inflammatory responses. RTA 402 and otherAIMs have been shown to inhibit pro-inflammatory NF-κB signaling in avariety of cell lines (Shishodia, 2006; Ahmad, 2006; Yore, 2006). Theeffect of RTA 408 on TNFα-induced NF-κB signaling was evaluated inHeLa/NF-κB-Luc cells, a human cervical adenocarcinoma cell line stablytransfected with a luciferase reporter construct under the control ofmultiple NF-κB transcriptional response elements. HeLa/NF-κB-Luc cellswere pretreated for 1 hour with RTA 408, followed by treatment with TNFα(10 ng/mL) for an additional 5 hours. After treatment, luminescence wasmeasured, and the effect of RTA 408 pretreatment on TNFα-inducedluciferase activity was determined. The average results and standarddeviations from three independent experiments are shown in FIG. 10. RTA408 dose-dependently inhibited TNFα-induced NF-κB activation with anIC₅₀ value of 517±83 nM. Similar results were observed in another NF-κBreporter cell line (A549/NF-κB-Luc) where RTA 408 inhibited TNFα-inducedNF-κB activation with an IC₅₀ value of 627 nM (range 614-649 nM). RTA408 was 1.6-1.8 fold more efficient at reducing expression from theNF-κB promoter reporter in HeLa/NF-κB-Luc cells than 63189 (854 nM) and63170 (953 nM), respectively.

The effect of RTA 408 on TNFα-induced phosphorylation of IκBα, a keystep in activation of the NF-κB pathway, was also evaluated in HeLacells. HeLa cells were pretreated with RTA 408 for 6 hours, followed bytreatment with TNFα (20 ng/mL) for 5 min. Total and phosphorylatedlevels of IκBα were evaluated by Western blot. Consistent with theresults from the luciferase reporter assay, RTA 408 inhibitedTNFα-induced phosphorylation of IκBα in a dose-dependent manner (FIG.11).

RTA 408 has also been demonstrated to inhibit other pro-inflammatorysignaling pathways, such as IL-6-induced signal transducer and activatorof transcription 3 (STAT3) phosphorylation and receptor activator ofNF-κB ligand (RANKL)-induced osteoclastogenesis. In HeLa cells,pretreatment with 1 μM RTA 408 for 6 hours inhibited phosphorylation ofSTAT3 induced by IL-6. Osteoclastogenesis is a multi-stepdifferentiation process that results from the binding of RANKL to itsreceptor, RANK, on cells of hematopoietic origin. This results in theactivation of NF-κB and MAPK, which in turn increase transcription ofosteoclast-specific target genes, including tartrate-resistant acidphosphatase (TRAP). The effect of RTA 408 on RANKL-inducedosteoclastogenesis was evaluated in the mouse macrophage cell lineRAW264.7. RAW264.7 cells were pretreated for 2 hours with RTA 408 andthen treated with 50 ng/mL recombinant mouse RANKL. RTA 408dose-dependently inhibited RANKL-induced TRAP activity and the formationof osteoclasts, with an IC₅₀ of ˜5-10 nM.

5. Effect of RTA 408 on Expression of Genes Encoding TransaminaseEnzymes

Transaminase elevations were observed in the 28-day toxicity studieswith RTA 408 in rats and, to a much lower extent, in monkeys. Similarfindings have been observed following oral administration of a relatedAIM (bardoxolone methyl) in humans (Pergola, 2011). One hypothesis forthis effect is that AIMs directly or indirectly increase transaminasegene expression in the absence of cellular toxicity. To assess whethertreatment with RTA 408 affects transaminase mRNA levels, mouse AML-12hepatocytes were treated with RTA 408 for 18 hours, and the mRNA levelsof genes encoding transaminases were measured using quantitative PCR.Treatment with RTA 408 increased mRNA levels of alanine transaminase 1(Alt1 or Gpt1) and aspartate transaminase 1 (Ast1 or Got1) (FIGS. 12a,c). RTA 408 had no effect on alanine transaminase 2 (Alt2 or Gpt2) mRNAlevels and reduced mRNA levels of aspartate transaminase 2 (Ast2 orGot2) (FIGS. 12b,d ). These results demonstrate that RTA 408, at theconcentrations tested (250 nM or 500 nM), affects transaminase geneexpression in vitro in a manner consistent with the effects of othercompounds in the AIM class. However, it is unclear how the results fromthis in vitro system at the RTA 408 concentrations tested relate to thepotential effects on transaminases at clinically-relevant dose levels inhumans.

6. Effect of RTA 408 on Levels of Glycolytic Intermediates

Studies in diabetic mice have demonstrated that bardoxolone methylincreases muscle-specific insulin-stimulated glucose uptake (Saha,2010). In humans, a higher percentage of patients receiving bardoxolonemethyl reported experiencing muscle cramps compared with patientsreceiving placebo (Pergola, 2011). Muscle spasms have also been reportedin diabetic patients following insulin administration, suggesting apossible association with muscle glucose metabolism. The effect of RTA408 on glycolytic metabolism was evaluated through the assessment oflactate and pyruvate levels in cultured rodent C2C12 muscle cells.Similar to treatment with insulin, treatment of differentiated C2C12myotubes with 1 μM or 2 μM RTA 408 for 3 hours significantly increasedintracellular and extracellular lactate levels in a dose-dependentmanner.

Treatment of C2C12 differentiated myotubes with 250 nM or 500 nM RTA 408for 18 hours also significantly (P<0.0001, noted by asterisks) increasedintracellular pyruvate levels in a dose-dependent manner (FIG. 13).Together, these results demonstrate that RTA 408, at the concentrationstested, can affect muscle glycolytic intermediates in vitro; however, itis unclear how the results from this in vitro system at the RTA 408concentrations tested relate to the potential effects on glucosemetabolism at clinically-relevant dose levels in humans.

7. In Vitro Evaluation of RTA 408 Efflux by MRP-1

The efflux ratio MRP-1 for RTA 408 (1.3) was experimentally determinedto be approximately ten-fold lower than 63170 (10) and 63171 (11.2) andover 40-fold lower than 63189 (57.1). The value determined for RTA 408indicates that it is not a substrate of MRP-1, whereas the othercompounds are.

C. Protective Effects of RTA 408 in Animal Models of Lung Disease

RTA 408 was tested in several animal models of pulmonary disease toevaluate its potential efficacy in the lung. For all studies, RTA 408was orally administered daily in sesame oil at dose levels in the rangeof 3 to 150 mg/kg. In most cases, RTA 408 was administered startingseveral days prior to the induction of the lung injury response.

1. LPS-induced Pulmonary Inflammation in Mice

RTA 408 was tested in two studies of LPS-induced pulmonary inflammationin mice. In the first study, intended to be a preliminary dose-rangefinder, RTA 408 (30, 100, or 150 mg/kg) was administered once daily for3 days, followed by LPS administration 1 hour after the final dose.Bronchoalveolar lavage fluid (BALF) was collected 20 hours after LPSadministration (21 hours after the final dose of RTA 408) and evaluatedfor levels of pro-inflammatory markers (i.e., IL-6, IL-12p40, TNF-α, andRANTES). RTA 408 treatment resulted in a significant reduction inIL-12p40 at all doses and in TNFα at the 100 and 150 mg/kg doses (FIG.14). In the second study, RTA 408 (10, 30, or 100 mg/kg) wasadministered daily for 6 days, followed by LPS administration 1 hourafter the final dose. In this study, significant decreases in bodyweight were observed at the 100 mg/kg dose level starting on Day 3.Significant reductions in TNFα were observed at the 10 mg/kg dose, andsignificant reductions in IL-12p40, TNFα, and RANTES were observed atthe 30 mg/kg dose (FIGS. 15a ). Further evaluation of lungs from mice inthis study revealed meaningful engagement of relevant Nrf2 target genes,including significant induction of NQO1 enzyme activity and increases intotal GSH at 10 and 30 mg/kg (FIG. 15b ).

8. Bleomycin-induced Pulmonary Fibrosis

The effect of RTA 408 was also evaluated in models of bleomycin-inducedpulmonary fibrosis in mice and rats. In the first preliminary study, RTA408 (10, 30, or 100 mg/kg) was administered to mice daily via oralgavage for 39 days, with bleomycin challenge (intranasal) on day 10. Onthe last day of dosing, lung tissue was collected and histology wasperformed to evaluate the extent of inflammation and interstitialfibrosis. In this model, no statistically significant effects wereobserved at the RTA 408 doses tested (FIGS. 16a & b). Additionalevaluation was performed using a rat model of pulmonary fibrosis thathas been extensively characterized at the Lovelace Respiratory ResearchInstitute. In this study, rats were challenged with bleomycin or salineby intratracheal administration on day 0. Following the challenge,animals received RTA 408 (3, 10, or 30 mg/kg) daily via oral gavage for28 days. Administration of the 30-mg/kg dose was stopped on day 14 dueto excessive dehydration and diarrhea in the animals. For the remaininganimals, bronchoalveolar lavage fluid was collected on day 28 forassessment of pro-inflammatory infiltrates, and lung tissue was analyzedfor hydroxyproline levels and histopathology. Challenge with bleomycinsulfate induced a substantial release of neutrophils and an increase insoluble collagen in the BALF, as well as an increase in hydroxyprolinein the lung. Treatment with 3 and 10 mg/kg RTA 408 significantlysuppressed polymorphonuclear (PMN) cell infiltration into the lungs andalso produced a meaningful reduction (˜10%-20%) in hydroxyprolinedeposition (FIGS. 17a & b).

Importantly, histopathological evaluation revealed a significantdecrease in collagen deposition, as assessed by trichrsome staining, inrats treated with RTA 408. Whereas bleomycin control animals primarilyexhibited moderate staining, animals treated with 10 mg/kg RTA 408 hadpredominantly minimal to mild staining (Table 2).

TABLE 2 Effect of RTA 408 on collagen deposition in rat lung as assessedby intensity of trichrome staining RTA 408 RTA 408 StainingIntensity^(a) Bleomycin Control (3 mg/kg) (10 mg/kg) Minimal 0 0 3 Mild1 0 4 Moderate 7 7 1 ^(a)Values represent intensity of staining inanimals with interstitial trichrome staining in areas ofbleomycin-induced lung alterations.

Further evaluation of lungs from rats in this study also revealedmeaningful engagement of relevant Nrf2 target genes (FIG. 18). RTA 408significantly and dose-dependently increased NQO1, Txnrd, Gsr, and Gstenzyme activity in the lungs of rats exposed to bleomycin, demonstratingNrf2 activation by RTA 408 in this disease setting.

9. Cigarette Smoke-induced COPD in Mice

RTA 408 was also tested in a mouse model of cigarette smoke-inducedCOPD. Mice received RTA 408 (3, 10, or 30 mg/kg) daily via oral gavagefor two weeks and were exposed to cigarette smoke five days per weekduring the RTA 408 dosing period. At the end of the study, lung tissueand BALF were collected for analysis of inflammatory infiltrates andcytokines. In this experiment, multiple-dose administration of RTA 408at doses as low as 3 mg/kg RTA 408 resulted in significant suppressionof pro-inflammatory cytokines, including KC (functional mouse homolog ofhuman IL-8) and TNFα. A summary of results from this study is presentedin FIGS. 19a-e . An AIM analog (63355) was tested in the same study forcomparison. 63355 is a compound of the formula:

Further evaluation of lungs from mice in this study also revealedmeaningful engagement of relevant Nrf2 target genes (FIG. 20). NQO1enzyme activity in the lung was significantly decreased by cigarettesmoke exposure; administration of RTA 408 rescued this loss. Txnrdenzyme activity was also induced by the 30 mg/kg dose of RTA 408. Ingeneral Gsr enzyme activity was not altered, and Gst enzyme activity wasdecreased with treatment—both of which were likely the consequence of atemporal response for these enzymes.

10. Ovalbumin-induced Asthma in Mice

The potential activity of RTA 408 was also evaluated in a pilot study ina mouse model of ovalbumin-induced asthma. Mice were sensitized with anIP injection of ovalbumin and aluminum hydroxide on Day 0 and Day 14 andchallenged intranasally with ovalbumin in saline on Days 14, 25, 26, and27. Mice received RTA 408 (3, 10, or 30 mg/kg) daily via oral gavage onDays 1-13 and 15-27. Following sensitization and challenge withovalbumin, vehicle-treated mice had a significant increase in the totalnumber of leukocytes compared with positive control(dexamethasone)-treated mice. An increase in the number of T cells and Bcells was also observed in the vehicle-treated mice. Treatment with RTA408 at 30 mg/kg significantly reduced the number and percentage of Bcells within the airways. RTA 408 (3 and 30 mg/kg) also significantlyreduced the number of macrophages, but not the mean percentage ofmacrophages, detected in the airways. These observations are suggestiveof potential efficacy in this model.

11. Effects of RTA 408 on LPS-induced Sepsis in Mice

Sepsis was induced on Day 0 with an IP injection of LPS (21 mg/kg), andsurvival was followed until Day 4. RTA 408 (10, 30, or 100 mg/kg) wasadministered daily via oral gavage from Day −2 to Day 2. In the vehiclecontrol group, 60% of the animals survived until Day 4 (higher than the˜40% survival rate expected in this model). In the RTA 408 treatmentgroups, 80% of the animals in the 10 mg/kg dose group and 90% of theanimals in the 30 mg/kg dose group survived until Day 4 (FIGS. 21c & d).For the 100 mg/kg dose group, 90% of the animals survived until Day 4,with only a single death occurring on Day 4. Although these RTA408-induced effects are indicative of profound efficacy in this model,the relatively high survival rate in the vehicle control group precludeda statistically-significant difference between the control and RTA408-treated groups. Results obtained using the compound RTA 405 are alsopresented (FIGS. 21a & b). RTA 405 is a compound of the formula:

12. Effects of RTA 408 against Radiation-Induced Oral Mucositis

Exposure to acute radiation directed to the buccal cheek pouch ofhamsters produces effects similar to those observed in oral ulcerativemucositis in humans. These effects include moderate to severe mucositischaracterized by severe erythema and vasodilation, erosion of thesuperficial mucosa, and formation of ulcers. A single study wasconducted to evaluate the effects of RTA 408 in this model. On Day 0,each hamster was given an acute radiation dose of 40 Gy directed to theleft buccal cheek pouch. RTA 408 (10, 30, or 100 mg/kg) was orallyadministered twice daily from Day −5 to Day −1, and Day 1 to Day 15.Beginning on Day 6 and continuing until Day 28 on alternate days, oralmucositis was evaluated using a standard 6-point scoring scale. Both the30 and 100 mg/kg doses of RTA 408 caused a significant reduction in theduration of ulcerative mucositis (FIG. 22). Furthermore, adose-dependent decrease in the percentage of animals with mucositisscores ≥3 was also observed. However, administration of RTA 408 at 30 or100 mg/kg caused significant dose-dependent reductions in weight gain inirradiated hamsters. Due to weight loss in excess of 20%, two out ofeight hamsters in the 100 mg/kg dose group were euthanized on Day 2.

13. Effect of RTA 408 on the Induction of Nrf2 Biomarkers in Vivo

As described above, a key molecular target of RTA 408 is Nrf2, a centraltranscriptional regulator of antioxidative cellular protection.Activation of Nrf2 induces upregulation of a battery of cytoprotectivegenes, including NQO1, enzymes involved in GSH synthesis [i.e.,glutamate-cysteine ligase catalytic and modifier subunits (Gclc andGclm)], enzymes involved in detoxification (i.e., glutathioneS-transferases [Gsts]), and efflux transporters [i.e., multidrugresistance—associated proteins (Mrps)]. Induction of these genes resultsin a coordinated cellular effort to protect against oxidative insult,highlighted by increased antioxidative capacity, induction ofglutathione synthesis, and conjugation and export of potentially harmfulmolecules from the cell. In addition to the efficacy endpoints and Nrf2target gene expression evaluated in the various animal models describedabove, the ability of RTA 408 to induce expression of Nrf2 target geneswas also assessed using tissues collected from healthy RTA 408-treatedmice, rats, and monkeys.

As part of the non-GLP 14-day toxicity studies of RTA 408 in mice, rats,and monkeys, tissues were collected for the purposes of measuring mRNAand enzyme activity levels of selected Nrf2 target genes. For mice andrats, liver samples were collected 4 hours after the final dose on Day14. For monkeys, blood (for PBMC isolation), liver, lung, and braintissue were collected 24 hours after the final dose on Day 14. Enzymeactivity for NQO1, Gst, and glutathione reductase (Gsr) were measured intissue homogenates. Levels of mRNA were determined using Quantigene Plex2.0 technology, which involves a hybridization-based assay using XMAP®LUMINEX® magnetic beads for direct quantification of mRNA targets. Inaddition, RTA 408 concentrations were measured in plasma and tissues byLC/MS/MS methods.

RTA 408 generally increased the expression of various Nrf2 target genesin a dose-dependent manner at doses of 10, 30, and 100 mg/kg (FIG. 23,FIG. 24a , FIGS. 25a & b). Transcriptional upregulation of Nrf2 targetgenes by RTA 408 also resulted in functional increases in theantioxidant response, as manifested by dose-dependent increases in NQO1,Gst, and Gsr enzyme activity in rodent liver, as well as monkey liverand lung (FIGS. 26a & b, FIGS. 27a & b, FIGS. 28a & b). Furthermore, inrodents liver exposure of RTA 408 correlated with the level of enzymeactivity of NQO1, the prototypical target gene for Nrf2 (FIG. 29b , FIG.30b ). In monkeys, the level of mRNA expression in PBMCs of both NQO1and sulfiredoxin 1 (SRXN1) correlated with plasma exposure to RTA 408(FIGS. 34a & b). Overall, RTA 408 increased mRNA levels and activity ofNrf2 targets, and such increases generally correlated with tissue andplasma exposures, suggesting Nrf2 targets may serve as feasiblebiomarkers for Nrf2 activation (FIGS. 31a & b) and may be useful forassessing pharmacological activity of RTA 408 in healthy human subjects.

D. Safety Pharmacology

A GLP-compliant safety pharmacology program was completed using RTA 408.This included in vitro and in vivo (monkey) studies on thecardiovascular system, as well as studies on the respiratory system andcentral nervous system in rats.

2. Evaluation of the Effects of RTA 408 on Cloned hERG ChannelsExpressed in HEK293 cells

This study was conducted to assess the effects of RTA 408 on the rapidlyactivating inward rectifying potassium current (I_(Kr)) conducted byhERG (human ether-a-go-go-related gene) channels stably expressed in thehuman embryonic kidney (HEK293) cell line. The effects of RTA 408 on thehERG-related potassium current were assessed using whole-cell patchclamp electrophysiology methods. RTA 408 was determined to have IC₅₀value of 12.4 μM in a hERG QPatch_Kv11.1 assay. This value was 2.5-3fold higher than the values for 63170 (4.9 μM) and 63189 (3.8respectively. The RTA 408 IC₅₀ value was similar to the 63171 value(15.7

3. Cardiovascular Evaluation of RTA 408 in the Cynomolgus Monkey

A single study was conducted to evaluate the potential cardiovasculareffects of RTA 408 in conscious freely moving cynomolgus monkeys. Thesame four male and four female cynomolgus monkeys were administered thevehicle (sesame oil) and RTA 408 at dose levels of 10, 30, and 100 mg/kgaccording to a Latin square design, with one animal/sex/treatment dosedeach week followed by a 14-day washout period between administrations,until each animal received all treatments. Vehicle and RTA 408 wereadministered to all animals via oral gavage at a dose volume of 5 mL/kg.

Animals were instrumented with telemetry transmitters for measurement ofbody temperature, blood pressure, heart rate, and electrocardiogram(ECG) evaluation. Body temperature, systolic, diastolic, and meanarterial blood pressure, heart rate, and ECG parameters (QRS durationand RR, PR, and QT intervals) were monitored continuously from at least2 hours pre-dose until at least 24 hours post-dose. ECG tracings wereprinted at designated time points from the cardiovascular monitoringdata and were qualitatively evaluated by a board-certified veterinarycardiologist. Prior to the first administration on study, untreatedanimals were continuously monitored for cardiovascular endpoints for atleast 24 hours, and these data were used in the calculation of thecorrected QT interval throughout the study.

Observations for morbidity, mortality, injury, and availability of foodand water were conducted at least twice daily for all animals. Clinicalobservations were conducted pre-dose, approximately 4 hours post-dose,and following completion of the cardiovascular monitoring period. Bodyweights were measured and recorded on the day prior to each treatmentadministration.

RTA 408 at dose levels of 10, 30, and 100 mg/kg did not producemortality, adverse clinical signs, or result in meaningful changes inbody weight (FIG. 32), body temperature, blood pressure, or qualitativeor quantitative (PR, RR, QRS, QT intervals) ECG parameters. In the 100mg/kg dose group, a small (1.6% on average) but statisticallysignificant increase in the corrected QT interval was observed; however,individual animal data did not show consistent increases in QTc thatwould indicate a test article related effect. Consequently, due to thesmall magnitude of change and lack of a consistent response inindividual animals, these slight increases in QTc were not considered tobe related to RTA 408 treatment. Therefore, oral administration of RTA408 produced no effects on cardiovascular function in cynomolgus monkeysat doses up to and including 100 mg/kg.

4. Neurobehavioral Evaluation of RTA 408 in Rats

The potential acute neurobehavioral toxicity of RTA 408 was evaluated inrats. Three treatment groups of 10 male and 10 female CD® [CRL:CD® (SD)]rats received RTA 408 at dose levels of 3, 10, or 30 mg/kg. Oneadditional group of 10 animals/sex served as the control and receivedvehicle (sesame oil). Vehicle or RTA 408 was administered to all groupsvia oral gavage once on Day 1 at a dose volume of 10 mL/kg.

Observations for morbidity, mortality, injury, and availability of foodand water were conducted twice daily for all animals. Observations forclinical signs were conducted prior to dosing on Day 1 and followingeach functional observational battery (FOB) evaluation. FOB evaluationswere conducted pre-dose (Day −1) and at approximately 4 and 24 hourspost-dose. Body weights were measured and recorded pre-dose on Day 1.

RTA 408 at doses of 3, 10, and 30 mg/kg did not produce mortality,adverse clinical observations, or effects on any of the neurobehavioralmeasures tested. Slight decreases in body weight gain were observedapproximately 24 hours after dosing in the 30 mg/kg group that maypotentially be test article-related. With respect to the basicneurobehavioral endpoints evaluated in this study, RTA 408 did notproduce any adverse effects in rats at doses up to and including 30mg/kg.

5. Pulmonary Evaluation of RTA 408 in Rats

The potential effect of RTA 408 on pulmonary function was evaluated inrats. Three treatment groups of eight male and eight female CD® [CRL:CD®(SD)] rats received RTA 408 at dose levels of 3, 10, or 30 mg/kg. Oneadditional group of eight animals/sex served as the control and receivedvehicle (sesame oil). Vehicle or RTA 408 was administered to all groupsvia oral gavage once on Day 1 at a dose volume of 10 mL/kg.

Observations for mortality, morbidity, injury, and availability of foodand water were conducted twice daily for all animals. Clinicalobservations were conducted prior to dosing, approximately 4 hourspost-dose, and following completion of the 8-hour pulmonary monitoringperiod. Body weights were measured and recorded on the day of RTA 408administration. Pulmonary function (respiratory rate, tidal volume, andminute volume) was monitored for at least 1 hour prior to dosing toestablish a baseline and for at least 8 hours post-dose.

RTA 408 at doses of 3, 10, and 30 mg/kg did not produce mortality,adverse clinical observations, or effects on any of the pulmonaryparameters evaluated. Therefore, with respect to the basic pulmonaryendpoints evaluated in this study, RTA 408 did not produce any adverseeffects in rats at doses up to and including 30 mg/kg.

E. Nonclinical Overview

1. Pharmacokinetics

RTA 408 has been investigated both in vitro and in vivo to assess its PKand metabolism properties. In vitro studies have been conducted todetermine RTA 408 plasma protein binding and blood/plasma partitioning,cytochrome P450 (CYP450) inhibition and induction, and to identifymetabolites formed by liver microsomes of mice, rats, monkeys, andhumans. Data pertaining to the in vivo absorption and distributionfollowing repeated administration of RTA 408 has been obtained primarilythrough monitoring of drug levels in plasma and select tissues fromtoxicology studies. Sensitive and selective liquid chromatography-massspectrometry-based bioanalytical methods (LC/MS/MS) have been used tomeasure concentrations of RTA 408 in plasma, blood, and tissues withappropriate accuracy and precision.

a. Absorption

The absorption and systemic pharmacokinetic behavior of RTA 408 wasstudied in mice, rats, and monkeys following single and repeated (daily)oral administration. Following oral administration of a suspensionformulation at doses of 10 to 100 mg/kg, maximal concentrations wereobserved within 1 to 2 hours in mice, and within 1 to 24 hours in ratsand monkeys. Systemic exposure to RTA 408 tended to be highest in rats,with lower levels observed in mice and monkeys. Estimates of theapparent terminal half-life of RTA 408 observed after oraladministration were generally in the 6- to 26-hour range, though theapparent prolonged absorption phase in some instances precludedcalculation of a definitive half-life estimate.

Systemic exposure to RTA 408 was generally similar in males and females.Exposure to RTA 408 following repeated daily oral administration tendedto be slightly higher (≤2-fold) than the exposure observed after asingle dose. Administration of RTA 408 over a dose range from 3 to 100mg/kg in a suspension formulation generally resulted indose-proportional increases in systemic exposure. However,administration of higher doses (100 to 800 mg/kg in monkeys; 500 to 2000mg/kg in rats) did not result in similar increases in exposure,suggesting saturation of absorption at doses above 100 mg/kg. Followingoral administration of an unoptimized (loose-filled) capsule formulationof RTA 408 (3 mg/kg) to monkeys, dose-normalized systemic exposuretended to be somewhat lower than that observed with a suspensionformulation.

The absorption and systemic pharmacokinetic behavior of RTA 408 wasstudied in rats using single and repeated topical administration. Theadministration of RTA 408 over a range of 0.01 to 3% showed lower plasmaconcentrations relative to similar oral dosing. The systemic exposure toRTA 408 generally increased in a dose dependent manner. The topicaladministration was formulated as a suspension in sesame oil.

Using rabbits, the ocular absorption and systemic pharmacokineticbehavior of RTA 408 was evaluated. RTA 408 was administered topically tothe eye once per day for 5 days. The ocular administration showed lowerplasma concentration of RTA 408 relative to when RTA 408 is administeredorally (FIG. 33). The amount of RTA 408 in the plasma even after fiveconsecutive days showed only a small change compared to theconcentration after the first dose relative to when RTA 408 wasadministered orally, where plasma concentrations were almost 100 foldhigher (FIG. 33).

b. Distribution

Plasma protein binding of RTA 408 was evaluated in mouse, rat, rabbit,dog, minipig, monkey, and human plasma at RTA 408 concentrations of10-2000 ng/mL using ultracentrifugation methodology. RTA 408 wasextensively bound to plasma proteins. Plasma protein binding in thenonclinical species ranged from 93% (mouse) to >99% (minipig), withbinding of 95% in the toxicology species (rat and monkey) and 97% inhuman. There was no evidence of concentration-dependent protein bindingin any species tested. Results from blood-to-plasma partitioningexperiments indicate that RTA 408 tended to distribute primarily in theplasma fraction of blood in a linear manner, with blood:plasma ratios<1.0 for all species and all concentrations tested.

The distribution of RTA 408 into tissues has been investigated afteroral administration to mice, rats, and monkeys. In the 14-day non-GLPtoxicity studies, select tissues (liver, lung, and brain) were collectedat a single time point (4 hours for rat and mouse; 24 hours for monkey)after the final dose of the study was administered and were analyzed forRTA 408 content using LC/MS/MS. RTA 408 readily distributes into lung,liver, and brain. In lung, RTA 408 concentrations at 4 hours in mice andrats were similar to or slightly higher (<2-fold) than concentrations inplasma, while at 24 hours in monkeys, RTA 408 concentrations in lungwere 6- to 16-fold higher than plasma concentrations. A similar patternwas observed for brain. In contrast, RTA 408 concentrations in liverwere 5- to 17-fold higher than plasma for mice and rats at 4 hours, and2- to 5-fold higher than plasma at 24 hours in monkeys.

The pharmacodynamic effects of RTA 408 in tissues were assessed in mice,rats, and monkeys, by monitoring the induction of Nrf2 target genes inthe same tissues collected for drug exposure from the 14-day toxicitystudies. Induction of Nrf2 target genes by RTA 408 resulted in increasesin the antioxidant response as manifested by dose-dependent increases inNQO1, glutathione S-transferase (Gst), and glutathione reductase (Gsr)enzyme activity in the examined tissues. Furthermore, in rodents, RTA408 liver content correlated with the level of enzyme activity for NQO1,the prototypical target gene for Nrf2. In monkeys, the level of mRNAexpression in peripheral blood mononuclear cells (PBMCs) for both NQO1and sulfiredoxin 1 (SRXN1) correlated with plasma exposure of RTA 408(FIGS. 34a & b). Overall, RTA 408 induced biomarkers of Nrf2 in rodentsand monkeys, and such inductions generally correlated well with tissueand plasma exposure to RTA 408.

When RTA 408 was administered to rabbits via ocular topicaladministration, the highest concentrations of the compound were found inthe cornea, retina, or iris while the vitreous humor, aqueous humor, andplasma showed significantly lower concentrations of RTA 408 (FIG. 35).

c. Metabolism

The metabolism of RTA 408 has been investigated after in vitroincubation of RTA 408 for 60 minutes with liver microsomes from mice,rats, monkeys, and humans in the presence of a nicotinamide adeninedinucleotide phosphate (NADPH)-regenerating system and a uridinediphosphate glucuronosyltransferase (UGT) reaction mixture. Extensiveturnover of RTA 408 was observed with primate microsomes, with <10% ofthe parent molecule remaining at the end of the 60-minute incubation inboth monkey and human microsomes. In contrast, the extent of metabolismwas lower in rodent microsomes, with >65% of the parent moleculeremaining at the end of the incubation. The lack of available authenticstandards for the various potential metabolites of RTA 408 precludedquantitative evaluation of the observed metabolites. From a qualitativeperspective, a similar pattern of RTA 408 metabolites was observedacross species, and included peaks with masses consistent with reductionand hydroxylation of RTA 408 as well as glucuronidation of RTA 408 or ofits reduction/hydroxylation metabolites. No unique human metaboliteswere observed, with all peaks in the human microsome incubations alsobeing observed in one or more of the preclinical species. In particular,based on in vitro microsome data, all human metabolites were present inrat or monkey, the selected rodent and non-rodent toxicity species.

d. Pharmacokinetic Drug Interactions

The potential for RTA 408 to inhibit cytochrome P450 (CYP450)-mediatedmetabolism was evaluated using pooled human liver microsomes andstandard substrates for specific CYP450 enzymes. RTA 408 directlyinhibited CYP2C8 and CYP3A4/5 with K_(i) values of approximately 0.5 μMfor each enzyme. No meaningful inhibition was observed for the otherenzymes tested (CYP1A2, CYP2B6, CYP2C9, CYP2C19, or CYP2D6), withinhibition <50% at the highest concentration tested (3 μM). In addition,there was little or no evidence of metabolism-dependent inhibition ofany of the enzymes tested. Future studies investigating the potentialfor CYP3A4/5-mediated drug-drug interactions may be warranted based onthese data, and the potentially high concentrations that may be achievedlocally in the gastrointestinal (GI) tract after oral administration.

The potential for RTA 408 to induce CYP450 enzyme expression wasevaluated using cultured human hepatocytes. Under conditions whereprototypical inducers caused the expected increases in CYP activity, RTA408 (up to 3 μM) was not an inducer of CYP1A2, CYP2B6, or CYP3A4 enzymeactivity in cultured human hepatocytes.

F. Effects of RTA 408 on Acute Radiation Dermatitis

The effects of RTA 408 as a topical or oral preventative for acuteradiation dermatitis have been examined. Using male BALB/c mice, a 30 Gydose of radiation was administered on day 0 (Table 3). The sesame oilvehicle or RTA 408 was administered to the rats on day −5 to −1 and days1 to 30. RTA 408 was administered both orally in 3, 10, and 30 mg/kg insesame oil and topically in percentage composition of 0.01, 0.1, and 1%in sesame oil. The dermatitis was blindly evaluated every other day fromday 4 to day 30. On day 12, the typical peak of dermatitis was observedand 4 mice were sacrificed 4 hours after administration of the dose. Theremaining mice were sacrificed on day 30 at 4 hours postdose. Plasma wascollected on days 12 and 30 as well as irradiated skin samples for mRNAand histological examination.

TABLE 3 Study Design for Acute Radiation Dermatitis Model Number ofRadiation Treatment Group Animals (Day 0) Treatment Schedule 1  9 males— Untreated — 2 10 males 30 Gy Untreated — 3 14 males 30 Gy VehicleControl Day −5 to −1 & (sesame oil) Day 1 to 30 4 14 males 30 Gy RTA408 - 0.01% or Day −5 to −1 & 3 mg/kg Day 1 to 30 5 14 males 30 Gy RTA408 - 0.1% or Day −5 to −1 & 10 mg/kg Day 1 to 30 6 14 males 30 Gy RTA408 - 1% or Day −5 to −1 & 30 mg/kg Day 1 to 30

In the test groups where the mice were treated with RTA 408, theincidence of dermatitis appeared to be slightly diminished in severitywhen RTA 408 was given in either an oral or topical administration(FIGS. 36-39). Furthermore, curves plotting the average dermatitisclinic score for the test groups as a function of time show some changewith the administration of RTA 408 either in oral or topical form fromthe untreated test groups (FIGS. 40-42) particularly in the case whereRTA 408 was given through an oral administration. Furthermore, as can beseen in Tables 4 and 5 below, the percentage of mice suffering fromdermatitis with a clinical score above 3 was significantly lower formice treated with RTA 408 through an oral administration while thepercentage of mice suffering from dermatitis with a clinical score above2 was slightly lower for test groups who were given a topicaladministration of RTA 408.

TABLE 4 Percentage of mice per testing group which scored above 2 intheir clinical dermatitis exam and given a topical treatment containingRTA 408 Day Day Day Day Day Day Day Day Day Day % animal- % animal- 1214 16 18 20 22 24 26 28 30 days >= 2 days >= 3 1 no radiation, untreated0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 2 irradiated, untreated0.0 50.0 83.3 83.3 83.3 100.0 66.7 50.0 50.0 50.0 35.6 0.0 3 irradiated,sesame oil 21.4 45.0 60.0 50.0 40.0 40.0 0.0 0.0 0.0 0.0 16.6 0.0 4irradiated, RTA 408- 0.01% 0.0 0.0 20.0 50.0 10.0 40.0 40.0 40.0 20.010.0 14.4 0.0 5 irradiated, RTA 408-0.1% 7.1 10.0 20.0 80.0 60.0 40.030.0 10.0 0.0 0.0 16.3 0.0 6 irradiated, RTA 408-1.0% 10.7 20.0 10.070.0 30.0 10.0 0.0 0.0 0.0 0.0 9.7 0.0

TABLE 5 Percentage of mice per testing group which scored above 3 intheir clinical dermatitis exam and given an oral treatment containingRTA 408 Day Day Day Day Day Day Day % animal- % animal- 16 18 20 22 2426 28 days >= 2 days >= 3 1 no radiation, untreated 0 0 0 0 0 0 0 0.00.0 2 irradiated, untreated 20 40 20 20 20 20 20 39.0 8.8 3 irradiated,sesame oil 35 50 40 30 20 0 0 45.6 10.9 4 irradiated, RTA 408-3 mg/kg 1010 0 0 0 0 0 32.5 1.3 5 irradiated, RTA 408-10 mg/kg 10 25 30 0 0 0 033.8 4.1 6 irradiated, RTA 408-30 mg/kg 10 20 10 0 0 0 0 28.8 2.5

G. Effects of RTA 408 on Fractionated Radiation Dermatitis

Utilizing RTA 408 through topical administration, the effects of RTA 408towards ameliorating the effects of fractionated radiation dermatitiswere measured. Using Balb/c mice, RTA 408 in a topical preparation wasadministered to the mice daily from day −5 to day 30 in three dosesranging from 0.01 to 1%. The mice were irradiated on days 0-2 and 5-7with six 10-Gy doses per day. Clinical dermatitis scores for the micewere evaluated blindly every two days from day 4 until the end of thestudy. In FIG. 43, the graph shows the change in the average clinicalscore for each group were plotted as a function of time. The graph showsa statistically significant improvement in the scores for mice treatedwith 0.1 to 1% topical formulations of RTA 408. Study and treatmentparameters can be found in Table 6.

TABLE 6 Study Conditions for Fractionated Radiation-Induced DermatitisRadiation Number of (Days 0-2, Treatment Group Animals 5-7) TreatmentSchedule 1  9 males — Untreated — 2 14 males 6x 10 Gy Untreated — 3 18males 6x 10 Gy Vehicle Control QD Days −5 to 30 (sesame oil) 4 18 males6x 10 Gy RTA 408 - 0.01% QD Days −5 to 30 5 18 males 6x 10 Gy RTA 408 -0.1% QD Days −5 to 30 6 18 males 6x 10 Gy RTA 408 - 1% QD Days −5 to 30By analyzing the average clinical scores that were shown in FIG. 43, anarea under the curve (AUC) analysis was performed, which yielded theseverity of the dermatitis relative to how long the dermatitispersisted. This AUC analysis allowed for direct comparison between thedifferent groups of mice and the effect of the different percentagecompositions of RTA 408 (FIG. 44 and Table 7). Administration of topicalRTA 408 formulations reduced Grade 2 and Grade 3 lesions from 60% and33% when the mice were only exposed to the vehicle to 21% and 6% withRTA 408 at 1%, concentration respectively. The other RTA compositionshowed some activity but was not as significant as that shown by the 1%formulation.

TABLE 7 Percentage of Dermatitis Score for Each Treatment Group Group %Days ≥ 2 % Days ≥ 3 No Rad, No Tx  0%  0% Rad, No Tx 66% 31% Rad, SesameOil 60% 33% Rad, RTA 408 (0.01%) 54% 29% Rad, RTA 408 (0.1%) 40% 13%Rad, RTA 408 (1%) 21%  6%

H. Effects of RTA 408 on a Model of Ocular Inflammation

A study of the effects of RTA 408 on ocular inflammation was carried outusing rabbits of the New Zealand albino strain. The rabbits were dividedinto 5 groups of 12 rabbits which were given three differentconcentrations of RTA 408 (0.01, 0.1, and 1%), VOLTARENE© collyre at0.1% and the vehicle (sesame oil). Each rabbit was given threeinstillations within 60 minutes before induction of paracentesis and twoinstillations within 30 minutes after induction of paracentesis. Eachinstillation was 50 μL and given in both eyes. Aqueous humor for 6animals per time-point was collected 30 minutes and again 2 hours afterinduction of paracentesis. The amount of inflammation was determined byprotein concentration in the aqueous humor. As shown in FIG. 45, RTA 408showed a reduction in aqueous humor protein similar to that of thehighest concentration of any of the other reference compounds (MAXIDEX®or mapracorat) at only 0.01% RTA 408 in the formulation. The effects ofincreasing concentration of RTA 408 appeared to be negligible as allconcentrations of RTA 408 appeared to show relatively similar effectswithin error in reducing aqueous humor protein concentration.

I. Polymorphs of RTA 408

RTA 408 Polymorphic Form A

Example 1: 17 g of RTA 408 was dissolved in 68 g of acetone. 620 g ofde-ionized water was added to a 500 mL jacketed reactor and cooled to 2°C. When the water was below 7° C., the RTA 408 solution was added to thereactor via an addition funnel. A slurry of solids formed. The slurrywas stirred in the reactor with nitrogen purge. Solids were isolatedusing vacuum filtration and dried under vacuum at room temperature togive Form A.

Example 2: 300 mg of RTA 408 was dissolved in 1 mL of ethyl acetate. Tothe clear solution, 2 mL of heptane was added. Crystallization occurredwithin 30 minutes. The slurry was stirred overnight and the solids wereisolated by vacuum filtration and dried at ambient temperature for 1hour. The solids were then dried in a vacuum oven at 50° C. overnight togive Form A.

Powder X-ray diffraction (PXRD) pattern and peak listing with relativeintensities are shown in FIG. 53 and Table 8, respectively. Differentialscanning calorimetry (DSC) and thermogravimetric analysis with massspectroscopy (TGA-MS) are shown in FIGS. 54 and 55, respectively.

The DSC of Form A indicated an essentially solvent free form with amelting point of 181.98° C. and enthalpy of fusion of 42.01 J/g. TheTGA-MS of Form A shows the loss of ˜0.5 wt.-% with traces of H₂O between25 and 200° C., predominantly above 160° C., indicating that RTA 408Polymorphic Form A may be slightly hygroscopic.

TABLE 8 Peak Listing of RTA 408 Form A Peak Position (°2θ) RelativeIntensity 10.601 11.0 11.638 7.1 12.121 4.6 13.021 10.9 13.435 100.015.418 12.7 15.760 5.9 17.830 19.7 18.753 38.3 19.671 7.5

RTA 408 Polymorphic Form B

Example 3: 1.0 g of RTA 408 was dissolved in 1.5 mL of acetone. In ascintillation vial, 10 mL of de-ionized water was heated to 50° C. andthe RTA 408 solution was added to the vial dropwise. Upon stirring for 2hours, a slurry of solids formed. The slurry was then cooled to roomtemperature. The resulting solids were isolated by filtration and driedin a vacuum oven at 50° C. overnight to give Form B.

Example 4: 2.9 g of RTA 408 was dissolved in 20 mL of isopropyl alcoholat reflux. 20 mL of heptane was added to the solution at reflux. Thesolution was cooled to room temperature and mixed for 1 hour. A slurryof solids formed. The solids were isolated by vacuum filtration anddried under vacuum at ambient temperature to give Form B.

Powder X-ray diffraction (PXRD) pattern and peak listing with relativeintensities are shown in FIG. 56 and Table 9, respectively. Differentialscanning calorimetry (DSC) and thermogravimetric analysis with massspectroscopy (TGA-MS) are shown in FIGS. 57 and 58, respectively.

The DSC of Form B indicated an essentially solvent free form with amelting point of 250.10° C. and enthalpy of fusion of 42.01 J/g. TheTGA-MS of Form B shows the slight loss of ˜0.2 wt.-% with traces of H₂Obetween 25 and 200° C., indicating that RTA 408 Polymorphic Form B maybe very slightly hygroscopic.

TABLE 9 Peak Listing of RTA 408 Form B Peak Position (°2θ) RelativeIntensity 7.552 4.2 10.339 100.0 11.159 48.4 12.107 80.7 14.729 35.215.329 11.4 15.857 8.4 16.824 11.3 17.994 10.9 18.344 4.1 19.444 10.419.764 10.2 20.801 5.7 22.414 10.1

J. Instrumental—Typical Measurement Conditions

Powder X-Ray Diffractometry (PXRD)

PXRD data were collected using a G3000 diffractometer (Inel Corp.,Artenay, France) equipped with a curved position sensitive detector andparallel beam optics. The diffractometer was operated with a copperanode tube (1.5 kW fine focus) at 40 kV and 30 mA. An incident beamgermanium monochromometer provided monochromatic radiation. Thediffractometer was calibrated using the attenuated direct beam atone-degree intervals. Calibration was checked using a silicon powderline position reference standard (NIST 640c). The instrument wascomputer controlled using the Symphonix software (Inel Corp., Artenay,France) and the data was analyzed using the Jade software (version9.0.4, Materials Data, Inc., Livermore, Calif.). The sample was loadedonto an aluminum sample holder and leveled with a glass slide.

Thermo Gravimetric Analysis/Mass Spectrometry

The TGA was run with TA instruments, data were collected on a thermalbalance (Q-5000, TA Instruments, New Castle, Del.) equipped with a dataanalyzer (Universal Analysis 2000, version 4.5A, TA Instruments, NewCastle, Del.). During experiments, the furnace was purged with nitrogenat 60 mL/minute, while the balance chamber was purged at 40 mL/minute.Temperature of the TGA furnace was calibrated using curie points ofaluminum and nickel. Sample size ranged from 2 to 20 mg, and a heatingrate of 10° C./minute was used.

For TGA-MS, the thermogravimetric analysis part was the same as above.The mass of evolved gas was analyzed with PFEIFFER GSD 301 T3 ThermoStar(PFEIFFER Vacuum, Asslar, Germany). The instrument was operated and dataevaluated with Software Quadstar 32-bit (V7.01, Inficon, LI-9496Balzers, Liechtenstein).

Differential Scanning calorimetery

A DSC (Q-2000, TA Instruments, New Castle, Del.) equipped with UniversalAnalysis 2000 software (Version 4.5A, TA Instruments, New Castle, Del.)was used to determine the DSC thermal traces. The temperature axis wascalibrated with biphenyl, indium, and tin standards. The cell constantwas calibrated with indium. Unless otherwise stated, the sample (2-5 mg)was encapsulated in a ventilated aluminum pan, and heated at a rate of10° C./minute under a nitrogen gas flow of 50 mL/minute during thestudy.

Abbreviations

Methods:

-   -   AUC area under the curve analysis    -   DSC differential scanning calorimetry    -   ¹H-NMR proton nuclear magnetic resonance spectroscopy    -   HPLC-MS high-performance liquid chromatography coupled to mass        spectroscopy    -   LC/MS/MS liquid chromatography-tandem mass spectrometry    -   PXRD powder X-ray diffraction    -   TGA-MS thermogravimetric analysis coupled to mass spectroscopy

Genes, Proteins, and Biological Parameters:

-   -   AIM antioxidant inflammation modulator    -   ARE antioxidant response element    -   ALP alkaline phosphatase    -   ALT alanine transaminase    -   ARE antioxidant response element    -   AST aspartate transaminase    -   AUC area under the curve    -   BAL bronchoalveolar lavage    -   BALF bronchoalveolar lavage fluid    -   COPD chronic obstructive pulmonary disease    -   COX-2 cyclooxygenase-2    -   Cr creatine    -   CYP450 cytochrome P450    -   Gclc glutamate-cysteine ligase, catalytic subunit    -   Gclm glutamate-cysteine ligase, modifier subunit    -   Glu glucose    -   GOT glutamic-oxaloacetic transaminase    -   GPT1 glutamic-pyruvate transaminase    -   GSH glutathione    -   GSR glutathione reductase    -   GST glutathione S-transferase    -   Gy Gray    -   H6PD hexose-6-phosphate dehydrogenase    -   hERG human ether a-go-go-related gene    -   HMOX1 heme oxygenase (decycling) 1    -   HO-1 heme oxygenase    -   IFN γ interferon-gamma    -   IL interleukin    -   iNOS inducible nitric oxide synthase    -   IκBα nuclear factor of kappa light polypeptide gene enhancer in        B-cells inhibitor, alpha    -   KC mouse IL-8 related protein    -   Keap1 Kelch-like ECH associated protein-1    -   LPS lipopolysaccharide    -   ME1 malic enzyme 1    -   MPCE micronucleated polychromatic erythrocytes    -   Mrps multidrug resistance-related proteins    -   NADPH nicotinamide adenine dinucleotide phosphate, reduced    -   NF-κB nuclear factor of kappa-light-chain-enhancer of activated        B cells    -   NO nitric oxide    -   NQO1 NAD(P)H quinone oxidoreductase 1    -   Nrf2 nuclear factor (erythroid-derived)-like 2    -   p-IκBα phosphorylated IκBα    -   PBMC peripheral blood mononuclear cell    -   PCE polychromatic erythrocytes    -   PGD phosphogluconate dehydrogenase    -   PMN polymorphonuclear    -   RANTES regulated and normal T cell expressed and secreted    -   SOD1 superoxide dismutase 1    -   SRXN1 sulfiredoxin-1    -   TG total glycerides    -   TKT transketolase    -   TNFα tumor necrosis factor alpha    -   TXNRD1 thioredoxin reductase 1

Miscellaneous:

-   -   min minute(s)    -   m.p. melting point    -   Ph phenyl    -   T temperature    -   wt.-% weight percent

K. Further Tables

TABLE 10 Parameters of FIG. 46 NOx Levels (% vs LPS Controls) Compound13 mg/kg 25 mg/kg 50 mg/kg RTA 405 * 44% 26% 18% 63415 30% 18% 16%

TABLE 11 63415: Primary In Vivo ADMET - Key Primary ADMET Assays andEndpoints Assay Key Endpoints 14-day mouse Tolerability, body weight,clinical chemistry toxicity Tissue distribution Nrf2 target gene mRNAexpression & enzyme activation in liver 14-day rat Tolerability, bodyweight, clinical chemistry, & limited toxicity histopathology Tissuedistribution and plasma TK Nrf2 target gene mRNA expression & enzymeactivation in liver 14-day monkey Tolerability, body weight, clinicalchemistry, & limited toxicity histopathology Tissue distribution andplasma TK Nrf2 target gene mRNA expression and enzyme activation inmultiple tissues & PBMCs

TABLE 12 Parameters of FIG. 49 Vehicle 63415 Dose (mg/kg) 0 10 30 100ALT (U/L) 100 39 63 91 AST (U/L) 156 98 147 167 ALP (U/L) 120 131 110 98Tot Bil (mg/dL) <0.2 <0.2 <0.2 <0.2 BUN (mg/dL) 17 15 15 15 Cr (mg/dL)<0.2 <0.2 <0.2 <0.2 Glu (mg/dL) 288 307 285 273

TABLE 13 63415 is Negative for Genotoxicity in the In Vivo MicronucleusStudy Number of Number of Treatment PCE/Total MPCE/ MPCE/ (n = 5/Erythrocytes Change from 1000 PCE PCE group) (Mean +/− SD) Control (%)(Mean +/− SD) Scored 24-h timepoint Sesame Oil 0.588 ± 0.04 — 0.2 ± 0.272/10000  125 mg/kg 0.543 ± 0.03  −8 0.3 ± 0.27 3/10000  250 mg/kg 0.520± 0.06 −12 0.3 ± 0.27 3/10000  500 mg/kg 0.426 ± 0.07 −28 0.0 ± 0.000/10000 1000 mg/kg 0.498 ± 0.05 −15 0.2 ± 0.27 2/10000 1500 mg/kg 0.499± 0.06 −15 0.4 ± 0.22 4/10000 2000 mg/kg 0.531 ± 0.05 −10 0.2 ± 0.272/10000 48-h timepoint Sesame Oil 0.526 ± 0.05 — 0.3 ± 0.27 3/10000  125mg/kg 0.453 ± 0.03 −14 0.2 ± 0.27 2/10000  250 mg/kg 0.391 ± 0.02 −260.2 ± 0.27 2/10000  500 mg/kg 0.339 ± 0.05 −36 0.3 ± 0.45 3/10000 1000mg/kg 0.344 ± 0.04 −35 0.1 ± 0.22 1/10000 1500 mg/kg 0.376 ± 0.05 −390.4 ± 0.42 4/10000 2000 mg/kg 0.360 ± 0.03 −32 0.1 ± 0.22 1/10000

TABLE 14 Parameters of FIG. 32 Tot ALT AST ALP Tot Bil BUN Cr ProtAlbumin Glucose Chol TG Treatment Day (U/L) (U/L) (U/L) (mg/dL) (mg/dL)(mg/dL) (g/dL) (g/dL) (mg/dL) (mg/dL) (mg/dL) Vehicle BL 30 29 320 0.1523 0.63 7.2 4.1 87 124 52 Day 14 37 37 345 0.23 18 0.63 6.9 4.1 63 13064 10 mg/kg BL 46 32 351 0.18 35 0.78 7.4 4 74 146 51 Day 14 46 38 3820.23 27 0.68 7.2 4 39 144 82 30 mg/kg BL 32 32 409 0.18 23 0.7 7.3 4.285 125 47 Day 14 47 43 416 0.2 20 0.58 7.2 4 53 122 64 100 mg/kg  BL 3235 381 0.15 24 0.7 6.9 4 96 137 37 Day 14 43 37 390 0.18 24 0.55 6 3.232 93 61

TABLE 15 In Vitro Activity of 63415 and 63355 63415 63355 NO IC50 (nM),RAW264.7 4.0 ± 1  0.63 ± 0.06 WST-1 IC50 (nM), RAW264.7 125 150 NQO1-ARE(fold at 62.5 nM in HuH7) 5.3 ± 1.0 6.5 ± 0.9

TABLE 16 Parameters of FIG. 47 Compound Plasma Whole Blood Brain LiverLung Kidney RTA 405 (nM) 130 1165 93 1143 1631 2357 63415 (nM) 51 6791081 985 533 1604

TABLE 17 Parameters of FIG. 48 Compound Liver Lung Kidney RTA 405 1.931.48 8.25 63415 10.9 1.75 10.9

All of the compounds, polymorphs, formulations, and methods disclosedand claimed herein can be made and executed without undueexperimentation in light of the present disclosure. While the compounds,polymorphs, formulations, and methods of this invention have beendescribed in terms of preferred embodiments, it will be apparent tothose of skill in the art that variations may be applied to thecompounds, polymorphs, formulations, and methods, as well as in thesteps or in the sequence of steps of the method described herein withoutdeparting from the concept, spirit, and scope of the invention. Morespecifically, it will be apparent that certain agents which are bothchemically and physiologically related may be substituted for the agentsdescribed herein while the same or similar results would be achieved.All such similar substitutes and modifications apparent to those skilledin the art are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

REFERENCES

The following references to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

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1-81. (canceled)
 82. A method of treating a condition associated withinflammation or oxidative stress in a patient in need thereof,comprising administering to the patient a therapeutically effectiveamount of the pharmaceutical composition comprising an active ingredientcomprising a polymorphic form of a compound having the formula:

wherein the polymorphic form is crystalline, having an X-ray powderdiffraction pattern (CuKα) comprising peaks at about (a) 10.601, 11.638,12.121, 13.021, 13,435, 15.418, 15.760, 17.830, 18.753, and 19.671 °2θ,or (b) 7.552, 10.339, 11.159, 12.107, 14.729, 15.329, 15.857, 16.824,17.994, 18.344, 19.444, 19.764, 20.801, and 22.414 °2θ; and apharmaceutically acceptable carrier, wherein the condition is a skindisease or disorder.
 83. The method of claim 82, wherein the skindisease or disorder is dermatitis, a thermal or chemical burn, a chronicwound, acne, alopecia, other disorders of the hair follicle,epidermolysis bullosa, sunburn, complications of sunburn, a disorder ofskin pigmentation, an aging-related skin condition, a post-surgicalwound, a scar from a skin injury or burn, psoriasis, a dermatologicalmanifestation of an autoimmune disease or a graft-versus host disease,skin cancer, a disorder involving hyperproliferation of skin cells. 84.The method of claim 82, wherein the skin disease or disorder isdermatitis.
 85. The method of claim 84, wherein the dermatitis isallergic dermatitis, atopic dermatitis, dermatitis due to chemicalexposure, or radiation-induced dermatitis.
 86. The method of claim 82,wherein the skin disease or disorder is a chronic wound.
 87. The methodof claim 86, wherein the chronic wound is a diabetic ulcer, a pressuresore, or a venous ulcer.
 88. The method of claim 82, wherein the skindisease or disorder is alopecia.
 89. The method of claim 88, wherein thealopecia is selected from baldness and drug-induced alopecia.
 90. Themethod of claim 82, wherein the skin disease or disorder is a disorderof skin pigmentation.
 91. The method of claim 90, wherein the disorderof skin pigmentation is vitiligo.
 92. The method of claim 82, whereinthe skin disease or disorder is a disorder involving hyperproliferationof skin cells.
 93. The method of claim 92, wherein the disorderinvolving hyperproliferation of skin cells is hyperkeratosis.
 94. Themethod of claim 82, wherein the skin disease or disorder is psoriasis.95. The method of claim 82, wherein the skin disease or disorder isassociated with exposure to radiation.
 96. The method of claim 95,wherein the radiation exposure leads to dermatitis.
 97. The method ofclaim 95, wherein the radiation exposure is acute.
 98. The method ofclaim 95, wherein the radiation exposure is fractionated.
 99. The methodof claim 82, wherein the pharmaceutical composition is administered in asingle dose per day.
 100. The method of claim 82, wherein thepharmaceutical composition is administered in more than one dose perday.
 101. The method of claim 82, wherein the pharmaceutical compositionis administered topically.
 102. The method of claim 101, wherein thetopical administration is administered to the skin.
 103. The method ofclaim 82, wherein the pharmaceutical composition is formulated as alotion, a cream, a gel, an oil, an ointment, a salve, or a suspension.104. The method of claim 82, wherein the amount of the active ingredientis from 0.01% to 5% by weight.